• [ 2023 ]
• [ 2021 ]
• [ 2020 ]
• [ 2019 ]
• [ 2018 ]
• [ 2017 ]
• [ 2016 ]
• [ 2015 ]
• [ 2014 ]
• [ 2013 ]
• [ 2012 ]
• [ 2011 ]
• [ 2010 ]
• [ 2009 ]
• [ 2008 ]
• [ 2007 ]
• [ 2006 ]
• [ 2005 ]
• [ 2004 ]
• [ 2003 ]
• [ 2002 ]

• Domain swap facilitates structural transitions of spider silk protein C-terminal domains. Rat, Charlotte; Heindl, Cedric; Neuweiler, Hannes. In Protein Science, 32(11), S. e4783-. John Wiley & Sons, Ltd, 2023.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.

  @article{rat2023domain, abstract = {Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.}, author = {Rat, Charlotte and Heindl, Cedric and Neuweiler, Hannes}, booktitle = {Protein Science}, journal = {Protein Science}, keywords = {hannes}, month = 11, number = 11, pages = {e4783–}, publisher = {John Wiley & Sons, Ltd}, title = {Domain swap facilitates structural transitions of spider silk protein C-terminal domains}, volume = 32, year = 2023 }

  %0 Journal Article %1 rat2023domain %A Rat, Charlotte %A Heindl, Cedric %A Neuweiler, Hannes %B Protein Science %D 2023 %I John Wiley & Sons, Ltd %J Protein Science %N 11 %P e4783-- %R https://doi.org/10.1002/pro.4783 %T Domain swap facilitates structural transitions of spider silk protein C-terminal domains %U https://doi.org/10.1002/pro.4783 %V 32 %X Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.
• Domain swap facilitates structural transitions of spider silk protein C-terminal domains. Rat, Charlotte; Heindl, Cedric; Neuweiler, Hannes. In Protein Science, 32(11), S. e4783. 2023.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Abstract Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.

  @article{https://doi.org/10.1002/pro.4783, abstract = {Abstract Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.}, author = {Rat, Charlotte and Heindl, Cedric and Neuweiler, Hannes}, journal = {Protein Science}, keywords = {hannes}, number = 11, pages = {e4783}, title = {Domain swap facilitates structural transitions of spider silk protein C-terminal domains}, volume = 32, year = 2023 }

  %0 Journal Article %1 https://doi.org/10.1002/pro.4783 %A Rat, Charlotte %A Heindl, Cedric %A Neuweiler, Hannes %D 2023 %J Protein Science %N 11 %P e4783 %R https://doi.org/10.1002/pro.4783 %T Domain swap facilitates structural transitions of spider silk protein C-terminal domains %U https://onlinelibrary.wiley.com/doi/abs/10.1002/pro.4783 %V 32 %X Abstract Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.

• Warhead Reactivity Limits the Speed of Inhibition of the Cysteine Protease Rhodesain. Johe, Patrick; Jung, Sascha; Endres, Erik; Kersten, Christian; Zimmer, Collin; Ye, Weixiang; Sönnichsen, Carsten; Hellmich, Ute A.; Sotriffer, Christoph; Schirmeister, Tanja; Neuweiler, Hannes. In ACS Chem. Biol., 16(4), S. 661–670. American Chemical Society, 2021.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.

  @article{johe2021warhead, abstract = {Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.}, author = {Johe, Patrick and Jung, Sascha and Endres, Erik and Kersten, Christian and Zimmer, Collin and Ye, Weixiang and Sönnichsen, Carsten and Hellmich, Ute A. and Sotriffer, Christoph and Schirmeister, Tanja and Neuweiler, Hannes}, booktitle = {ACS Chemical Biology}, journal = {ACS Chem. Biol.}, keywords = {hannes}, month = {04}, number = 4, pages = {661–670}, publisher = {American Chemical Society}, title = {Warhead Reactivity Limits the Speed of Inhibition of the Cysteine Protease Rhodesain}, volume = 16, year = 2021 }

  %0 Journal Article %1 johe2021warhead %A Johe, Patrick %A Jung, Sascha %A Endres, Erik %A Kersten, Christian %A Zimmer, Collin %A Ye, Weixiang %A Sönnichsen, Carsten %A Hellmich, Ute A. %A Sotriffer, Christoph %A Schirmeister, Tanja %A Neuweiler, Hannes %B ACS Chemical Biology %D 2021 %I American Chemical Society %J ACS Chem. Biol. %N 4 %P 661--670 %R 10.1021/acschembio.0c00911 %T Warhead Reactivity Limits the Speed of Inhibition of the Cysteine Protease Rhodesain %U https://doi.org/10.1021/acschembio.0c00911 %V 16 %X Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.
• Structure, interdomain dynamics, and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes. Johé, Patrick; Jaenicke, Elmar; Neuweiler, Hannes; Schirmeister, Tanja; Kersten, Christian; Hellmich, Ute A. In Journal of Biological Chemistry, 296, S. 100565-. 2021.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood–brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases.

  @article{johe2021structure, abstract = {Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood–brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases.}, author = {Johé, Patrick and Jaenicke, Elmar and Neuweiler, Hannes and Schirmeister, Tanja and Kersten, Christian and Hellmich, Ute A.}, journal = {Journal of Biological Chemistry}, keywords = {hannes}, pages = {100565–}, title = {Structure, interdomain dynamics, and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes}, volume = 296, year = 2021 }

  %0 Journal Article %1 johe2021structure %A Johé, Patrick %A Jaenicke, Elmar %A Neuweiler, Hannes %A Schirmeister, Tanja %A Kersten, Christian %A Hellmich, Ute A. %D 2021 %J Journal of Biological Chemistry %P 100565-- %R https://doi.org/10.1016/j.jbc.2021.100565 %T Structure, interdomain dynamics, and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes %U https://www.sciencedirect.com/science/article/pii/S0021925821003434 %V 296 %X Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood–brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases.
• Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics. Schubert, Jonathan; Schulze, Andrea; Prodromou, Chrisostomos; Neuweiler, Hannes. In Nature Communications, 12(1), S. 6964-. 2021.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.

  @article{schubert2021twocolour, abstract = {Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.}, author = {Schubert, Jonathan and Schulze, Andrea and Prodromou, Chrisostomos and Neuweiler, Hannes}, journal = {Nature Communications}, keywords = {hannes}, number = 1, pages = {6964–}, title = {Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics}, volume = 12, year = 2021 }

  %0 Journal Article %1 schubert2021twocolour %A Schubert, Jonathan %A Schulze, Andrea %A Prodromou, Chrisostomos %A Neuweiler, Hannes %D 2021 %J Nature Communications %N 1 %P 6964-- %R 10.1038/s41467-021-27286-5 %T Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics %U https://doi.org/10.1038/s41467-021-27286-5 %V 12 %X Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.
• Allosteric coupling of sub-millisecond clamshell motions in ionotropic glutamate receptor ligand-binding domains. Rajab, Suhaila; Bismin, Leah; Schwarze, Simone; Pinggera, Alexandra; Greger, Ingo H.; Neuweiler, Hannes. In Communications Biology, 4(1), S. 1056-. 2021.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Ionotropic glutamate receptors (iGluRs) mediate signal transmission in the brain and are important drug targets. Structural studies show snapshots of iGluRs, which provide a mechanistic understanding of gating, yet the rapid motions driving the receptor machinery are largely elusive. Here we detect kinetics of conformational change of isolated clamshell-shaped ligand-binding domains (LBDs) from the three major iGluR sub-types, which initiate gating upon binding of agonists. We design fluorescence probes to measure domain motions through nanosecond fluorescence correlation spectroscopy. We observe a broad kinetic spectrum of LBD dynamics that underlie activation of iGluRs. Microsecond clamshell motions slow upon dimerization and freeze upon binding of full and partial agonists. We uncover allosteric coupling within NMDA LBD hetero-dimers, where binding of L-glutamate to the GluN2A LBD stalls clamshell motions of the glycine-binding GluN1 LBD. Our results reveal rapid LBD dynamics across iGluRs and suggest a mechanism of negative allosteric cooperativity in NMDA receptors.

  @article{rajab2021allosteric, abstract = {Ionotropic glutamate receptors (iGluRs) mediate signal transmission in the brain and are important drug targets. Structural studies show snapshots of iGluRs, which provide a mechanistic understanding of gating, yet the rapid motions driving the receptor machinery are largely elusive. Here we detect kinetics of conformational change of isolated clamshell-shaped ligand-binding domains (LBDs) from the three major iGluR sub-types, which initiate gating upon binding of agonists. We design fluorescence probes to measure domain motions through nanosecond fluorescence correlation spectroscopy. We observe a broad kinetic spectrum of LBD dynamics that underlie activation of iGluRs. Microsecond clamshell motions slow upon dimerization and freeze upon binding of full and partial agonists. We uncover allosteric coupling within NMDA LBD hetero-dimers, where binding of L-glutamate to the GluN2A LBD stalls clamshell motions of the glycine-binding GluN1 LBD. Our results reveal rapid LBD dynamics across iGluRs and suggest a mechanism of negative allosteric cooperativity in NMDA receptors.}, author = {Rajab, Suhaila and Bismin, Leah and Schwarze, Simone and Pinggera, Alexandra and Greger, Ingo H. and Neuweiler, Hannes}, journal = {Communications Biology}, keywords = {hannes}, number = 1, pages = {1056–}, title = {Allosteric coupling of sub-millisecond clamshell motions in ionotropic glutamate receptor ligand-binding domains}, volume = 4, year = 2021 }

  %0 Journal Article %1 rajab2021allosteric %A Rajab, Suhaila %A Bismin, Leah %A Schwarze, Simone %A Pinggera, Alexandra %A Greger, Ingo H. %A Neuweiler, Hannes %D 2021 %J Communications Biology %N 1 %P 1056-- %R 10.1038/s42003-021-02605-0 %T Allosteric coupling of sub-millisecond clamshell motions in ionotropic glutamate receptor ligand-binding domains %U https://doi.org/10.1038/s42003-021-02605-0 %V 4 %X Ionotropic glutamate receptors (iGluRs) mediate signal transmission in the brain and are important drug targets. Structural studies show snapshots of iGluRs, which provide a mechanistic understanding of gating, yet the rapid motions driving the receptor machinery are largely elusive. Here we detect kinetics of conformational change of isolated clamshell-shaped ligand-binding domains (LBDs) from the three major iGluR sub-types, which initiate gating upon binding of agonists. We design fluorescence probes to measure domain motions through nanosecond fluorescence correlation spectroscopy. We observe a broad kinetic spectrum of LBD dynamics that underlie activation of iGluRs. Microsecond clamshell motions slow upon dimerization and freeze upon binding of full and partial agonists. We uncover allosteric coupling within NMDA LBD hetero-dimers, where binding of L-glutamate to the GluN2A LBD stalls clamshell motions of the glycine-binding GluN1 LBD. Our results reveal rapid LBD dynamics across iGluRs and suggest a mechanism of negative allosteric cooperativity in NMDA receptors.

• NMR assignments of a dynamically perturbed and dimerization inhibited N-terminal domain variant of a spider silk protein from E. australis. Goretzki, Benedikt; Heiby, Julia C.; Hacker, Carolin; Neuweiler, Hannes; Hellmich, Ute A. In Biomolecular NMR Assignments, 14(1), S. 67–71. 2020.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Web spiders use specialized glands to produce silk proteins, so-called spidroins, which assemble into extraordinarily tough silk fibers through tightly regulated phase and structural transitions. A crucial step in the polymerization of spidroins is the pH-triggered assembly of their N-terminal domains (NTDs) into tight dimers. Major ampullate spidroin NTDs contain an unusually high content of the amino acid methionine. We previously showed that the simultaneous mutation of the six hydrophobic core methionine residues to leucine in the NTD of the major ampullate spidroin 1 from Euprosthenops australis, a nursery web spider, yields a protein (L6-NTD) retaining a three-dimensional fold identical to the wildtype (WT) domain, yet with a significantly increased stability. Further, the dynamics of the L6-NTD are significantly reduced and the ability to dimerize is severely impaired compared to the WT domain. These properties lead to significant changes in the NMR spectra between WT and L6-NTD so that the previously available WT-NTD assignments cannot be transferred to the mutant protein. Here, we thus report the de novo NMR backbone and side chain assignments of the major ampullate spidroin 1 L6-NTD variant from E. australis as a prerequisite for obtaining further insights into protein structure and dynamics.

  @article{goretzki2020assignments, abstract = {Web spiders use specialized glands to produce silk proteins, so-called spidroins, which assemble into extraordinarily tough silk fibers through tightly regulated phase and structural transitions. A crucial step in the polymerization of spidroins is the pH-triggered assembly of their N-terminal domains (NTDs) into tight dimers. Major ampullate spidroin NTDs contain an unusually high content of the amino acid methionine. We previously showed that the simultaneous mutation of the six hydrophobic core methionine residues to leucine in the NTD of the major ampullate spidroin 1 from Euprosthenops australis, a nursery web spider, yields a protein (L6-NTD) retaining a three-dimensional fold identical to the wildtype (WT) domain, yet with a significantly increased stability. Further, the dynamics of the L6-NTD are significantly reduced and the ability to dimerize is severely impaired compared to the WT domain. These properties lead to significant changes in the NMR spectra between WT and L6-NTD so that the previously available WT-NTD assignments cannot be transferred to the mutant protein. Here, we thus report the de novo NMR backbone and side chain assignments of the major ampullate spidroin 1 L6-NTD variant from E. australis as a prerequisite for obtaining further insights into protein structure and dynamics.}, author = {Goretzki, Benedikt and Heiby, Julia C. and Hacker, Carolin and Neuweiler, Hannes and Hellmich, Ute A.}, journal = {Biomolecular NMR Assignments}, keywords = {hannes}, number = 1, pages = {67–71}, title = {NMR assignments of a dynamically perturbed and dimerization inhibited N-terminal domain variant of a spider silk protein from E. australis}, volume = 14, year = 2020 }

  %0 Journal Article %1 goretzki2020assignments %A Goretzki, Benedikt %A Heiby, Julia C. %A Hacker, Carolin %A Neuweiler, Hannes %A Hellmich, Ute A. %D 2020 %J Biomolecular NMR Assignments %N 1 %P 67--71 %R 10.1007/s12104-019-09922-w %T NMR assignments of a dynamically perturbed and dimerization inhibited N-terminal domain variant of a spider silk protein from E. australis %U https://doi.org/10.1007/s12104-019-09922-w %V 14 %X Web spiders use specialized glands to produce silk proteins, so-called spidroins, which assemble into extraordinarily tough silk fibers through tightly regulated phase and structural transitions. A crucial step in the polymerization of spidroins is the pH-triggered assembly of their N-terminal domains (NTDs) into tight dimers. Major ampullate spidroin NTDs contain an unusually high content of the amino acid methionine. We previously showed that the simultaneous mutation of the six hydrophobic core methionine residues to leucine in the NTD of the major ampullate spidroin 1 from Euprosthenops australis, a nursery web spider, yields a protein (L6-NTD) retaining a three-dimensional fold identical to the wildtype (WT) domain, yet with a significantly increased stability. Further, the dynamics of the L6-NTD are significantly reduced and the ability to dimerize is severely impaired compared to the WT domain. These properties lead to significant changes in the NMR spectra between WT and L6-NTD so that the previously available WT-NTD assignments cannot be transferred to the mutant protein. Here, we thus report the de novo NMR backbone and side chain assignments of the major ampullate spidroin 1 L6-NTD variant from E. australis as a prerequisite for obtaining further insights into protein structure and dynamics.

• On-target restoration of a split T cell-engaging antibody for precision immunotherapy. Banaszek, Agnes; Bumm, Thomas G. P.; Nowotny, Boris; Geis, Maria; Jacob, Kim; Wölfl, Matthias; Trebing, Johannes; Kucka, Kirstin; Kouhestani, Dina; Gogishvili, Tea; Krenz, Bastian; Lutz, Justina; Rasche, Leo; Hönemann, Dirk; Neuweiler, Hannes; Heiby, Julia C.; Bargou, Ralf C.; Wajant, Harald; Einsele, Hermann; Riethmüller, Gert; Stuhler, Gernot. In Nature Communications, 10(1), S. 5387-. 2019.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.

  @article{banaszek2019ontarget, abstract = {T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.}, author = {Banaszek, Agnes and Bumm, Thomas G. P. and Nowotny, Boris and Geis, Maria and Jacob, Kim and Wölfl, Matthias and Trebing, Johannes and Kucka, Kirstin and Kouhestani, Dina and Gogishvili, Tea and Krenz, Bastian and Lutz, Justina and Rasche, Leo and Hönemann, Dirk and Neuweiler, Hannes and Heiby, Julia C. and Bargou, Ralf C. and Wajant, Harald and Einsele, Hermann and Riethmüller, Gert and Stuhler, Gernot}, journal = {Nature Communications}, keywords = {hannes}, number = 1, pages = {5387–}, title = {On-target restoration of a split T cell-engaging antibody for precision immunotherapy}, volume = 10, year = 2019 }

  %0 Journal Article %1 banaszek2019ontarget %A Banaszek, Agnes %A Bumm, Thomas G. P. %A Nowotny, Boris %A Geis, Maria %A Jacob, Kim %A Wölfl, Matthias %A Trebing, Johannes %A Kucka, Kirstin %A Kouhestani, Dina %A Gogishvili, Tea %A Krenz, Bastian %A Lutz, Justina %A Rasche, Leo %A Hönemann, Dirk %A Neuweiler, Hannes %A Heiby, Julia C. %A Bargou, Ralf C. %A Wajant, Harald %A Einsele, Hermann %A Riethmüller, Gert %A Stuhler, Gernot %D 2019 %J Nature Communications %N 1 %P 5387-- %R 10.1038/s41467-019-13196-0 %T On-target restoration of a split T cell-engaging antibody for precision immunotherapy %U https://doi.org/10.1038/s41467-019-13196-0 %V 10 %X T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.
• Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk. Heiby, Julia C.; Goretzki, Benedikt; Johnson, Christopher M.; Hellmich, Ute A.; Neuweiler, Hannes. In Nature Communications, 10(1), S. 4378-. 2019.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider’s dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function.

  @article{heiby2019methionine, abstract = {Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider’s dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function.}, author = {Heiby, Julia C. and Goretzki, Benedikt and Johnson, Christopher M. and Hellmich, Ute A. and Neuweiler, Hannes}, journal = {Nature Communications}, keywords = {hannes}, number = 1, pages = {4378–}, title = {Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk}, volume = 10, year = 2019 }

  %0 Journal Article %1 heiby2019methionine %A Heiby, Julia C. %A Goretzki, Benedikt %A Johnson, Christopher M. %A Hellmich, Ute A. %A Neuweiler, Hannes %D 2019 %J Nature Communications %N 1 %P 4378-- %R 10.1038/s41467-019-12365-5 %T Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk %U https://doi.org/10.1038/s41467-019-12365-5 %V 10 %X Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider’s dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function.
• The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation. Mercier, Rebecca; Wolmarans, Annemarie; Schubert, Jonathan; Neuweiler, Hannes; Johnson, Jill L.; LaPointe, Paul. In Nature Communications, 10(1), S. 1273-. 2019.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.

  @article{mercier2019conserved, abstract = {Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.}, author = {Mercier, Rebecca and Wolmarans, Annemarie and Schubert, Jonathan and Neuweiler, Hannes and Johnson, Jill L. and LaPointe, Paul}, journal = {Nature Communications}, keywords = {hannes}, number = 1, pages = {1273–}, title = {The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation}, volume = 10, year = 2019 }

  %0 Journal Article %1 mercier2019conserved %A Mercier, Rebecca %A Wolmarans, Annemarie %A Schubert, Jonathan %A Neuweiler, Hannes %A Johnson, Jill L. %A LaPointe, Paul %D 2019 %J Nature Communications %N 1 %P 1273-- %R 10.1038/s41467-019-09299-3 %T The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation %U https://doi.org/10.1038/s41467-019-09299-3 %V 10 %X Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.

• Two-step self-assembly of a spider silk molecular clamp. Rat, Charlotte; Heiby, Julia C.; Bunz, Jessica P.; Neuweiler, Hannes. In Nature Communications, 9(1), S. 4779-. 2018.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Web spiders synthesize silk fibers of unique strength and extensibility through the controlled self-assembly of protein building blocks, so-called spidroins. The spidroin C-terminal domain is highly conserved and connects two polypeptide chains through formation of an all-helical, intertwined dimer. Here we use contact-induced fluorescence self-quenching and resonance energy transfer in combination with far-UV circular dichroism spectroscopy as three orthogonal structural probes to dissect the mechanism of folding and dimerization of a spidroin C-terminal domain from the major ampullate gland of the nursery web spider Euprosthenops australis. We show that helices forming the dimer core assemble very rapidly and fold on association. Subsequently, peripheral helices fold and dock slowly onto the preformed core. Lability of outer helices facilitates formation of a highly expanded, partially folded dimer. The high end-to-end distance of chain termini in the partially folded dimer suggests an extensibility module that contributes to elasticity of spider silk.

  @article{rat2018twostep, abstract = {Web spiders synthesize silk fibers of unique strength and extensibility through the controlled self-assembly of protein building blocks, so-called spidroins. The spidroin C-terminal domain is highly conserved and connects two polypeptide chains through formation of an all-helical, intertwined dimer. Here we use contact-induced fluorescence self-quenching and resonance energy transfer in combination with far-UV circular dichroism spectroscopy as three orthogonal structural probes to dissect the mechanism of folding and dimerization of a spidroin C-terminal domain from the major ampullate gland of the nursery web spider Euprosthenops australis. We show that helices forming the dimer core assemble very rapidly and fold on association. Subsequently, peripheral helices fold and dock slowly onto the preformed core. Lability of outer helices facilitates formation of a highly expanded, partially folded dimer. The high end-to-end distance of chain termini in the partially folded dimer suggests an extensibility module that contributes to elasticity of spider silk.}, author = {Rat, Charlotte and Heiby, Julia C. and Bunz, Jessica P. and Neuweiler, Hannes}, journal = {Nature Communications}, keywords = {hannes}, number = 1, pages = {4779–}, title = {Two-step self-assembly of a spider silk molecular clamp}, volume = 9, year = 2018 }

  %0 Journal Article %1 rat2018twostep %A Rat, Charlotte %A Heiby, Julia C. %A Bunz, Jessica P. %A Neuweiler, Hannes %D 2018 %J Nature Communications %N 1 %P 4779-- %R 10.1038/s41467-018-07227-5 %T Two-step self-assembly of a spider silk molecular clamp %U https://doi.org/10.1038/s41467-018-07227-5 %V 9 %X Web spiders synthesize silk fibers of unique strength and extensibility through the controlled self-assembly of protein building blocks, so-called spidroins. The spidroin C-terminal domain is highly conserved and connects two polypeptide chains through formation of an all-helical, intertwined dimer. Here we use contact-induced fluorescence self-quenching and resonance energy transfer in combination with far-UV circular dichroism spectroscopy as three orthogonal structural probes to dissect the mechanism of folding and dimerization of a spidroin C-terminal domain from the major ampullate gland of the nursery web spider Euprosthenops australis. We show that helices forming the dimer core assemble very rapidly and fold on association. Subsequently, peripheral helices fold and dock slowly onto the preformed core. Lability of outer helices facilitates formation of a highly expanded, partially folded dimer. The high end-to-end distance of chain termini in the partially folded dimer suggests an extensibility module that contributes to elasticity of spider silk.

• Conservation of folding and association within a family of spidroin N-terminal domains. Heiby, Julia C.; Rajab, Suhaila; Rat, Charlotte; Johnson, Christopher M.; Neuweiler, Hannes. In Scientific Reports, 7(1), S. 16789. Nature Publishing Group, 2017.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Web spiders synthesize silk fibres, nature’s toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce.

  @article{heiby2017conservation, abstract = {Web spiders synthesize silk fibres, nature’s toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce.}, author = {Heiby, Julia C. and Rajab, Suhaila and Rat, Charlotte and Johnson, Christopher M. and Neuweiler, Hannes}, journal = {Scientific Reports}, keywords = {hannes}, month = 12, number = 1, pages = 16789, publisher = {Nature Publishing Group}, title = {Conservation of folding and association within a family of spidroin N-terminal domains}, volume = 7, year = 2017 }

  %0 Journal Article %1 heiby2017conservation %A Heiby, Julia C. %A Rajab, Suhaila %A Rat, Charlotte %A Johnson, Christopher M. %A Neuweiler, Hannes %D 2017 %I Nature Publishing Group %J Scientific Reports %N 1 %P 16789 %R 10.1038/s41598-017-16881-6 %T Conservation of folding and association within a family of spidroin N-terminal domains %U https://doi.org/10.1038/s41598-017-16881-6 %V 7 %X Web spiders synthesize silk fibres, nature’s toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce.

• Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism. Schulze, Andrea; Beliu, Gerti; Helmerich, Dominic A; Schubert, Jonathan; Pearl, Laurence H; Prodromou, Chrisostomos; Neuweiler, Hannes. In Nat Chem Biol, advance online publication. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2016.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.

  @article{schulze2016cooperation, abstract = {The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.}, author = {Schulze, Andrea and Beliu, Gerti and Helmerich, Dominic A and Schubert, Jonathan and Pearl, Laurence H and Prodromou, Chrisostomos and Neuweiler, Hannes}, journal = {Nat Chem Biol}, keywords = {hannes}, month = {06}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism}, volume = {advance online publication}, year = 2016 }

  %0 Journal Article %1 schulze2016cooperation %A Schulze, Andrea %A Beliu, Gerti %A Helmerich, Dominic A %A Schubert, Jonathan %A Pearl, Laurence H %A Prodromou, Chrisostomos %A Neuweiler, Hannes %D 2016 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Chem Biol %T Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism %U http://dx.doi.org/10.1038/nchembio.2111 %V advance online publication %X The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.

• β-Structure within the Denatured State of the Helical Protein Domain BBL. Thukral, Lipi; Schwarze, Simone; Daidone, Isabella; Neuweiler, Hannes. In Journal of Molecular Biology. 2015.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Abstract Protein denatured states are the origin of both healthy and toxic conformational species. Denatured states of ultrafast folding proteins are of interest in mechanistic studies because they are energetically close to the kinetic bottleneck of folding. However, their transient nature makes them elusive to experiment. Here, we generated the denatured state of the helical domain BBL that is poised to fold in microseconds by a single-point mutation and combined circular dichroism spectroscopy, single-molecule fluorescence fluctuation analysis, and computer simulation to characterize its structure and dynamics. Circular dichroism showed a largely unfolded ensemble with marginal helix but significant β-sheet content. Main-chain structure and dynamics were unaffected by side-chain interactions that stabilize the native state, as revealed by site-directed mutagenesis and nanosecond loop closure kinetics probed by fluorescence correlation spectroscopy. Replica-exchange and constant-temperature molecular dynamics simulations showed a highly collapsed, hydrogen-bonded denatured state containing turn and β-sheet structure and few nucleating helices in an otherwise unfolded ensemble. An irregular β-hairpin element that connects helices in the native fold was poised to be formed. The surprising observation of β-structure in regions that form helices in the native state is reconciled by a generic low-energy pathway from the northwest quadrant of Ramachandran space to the helical basin present under folding conditions, proposed recently. Our results show that, indeed, rapid nucleation of helix emanates from β-structure formed early within a collapsed ensemble of unfolded conformers.

  @article{thukralstructure, abstract = {Abstract Protein denatured states are the origin of both healthy and toxic conformational species. Denatured states of ultrafast folding proteins are of interest in mechanistic studies because they are energetically close to the kinetic bottleneck of folding. However, their transient nature makes them elusive to experiment. Here, we generated the denatured state of the helical domain BBL that is poised to fold in microseconds by a single-point mutation and combined circular dichroism spectroscopy, single-molecule fluorescence fluctuation analysis, and computer simulation to characterize its structure and dynamics. Circular dichroism showed a largely unfolded ensemble with marginal helix but significant β-sheet content. Main-chain structure and dynamics were unaffected by side-chain interactions that stabilize the native state, as revealed by site-directed mutagenesis and nanosecond loop closure kinetics probed by fluorescence correlation spectroscopy. Replica-exchange and constant-temperature molecular dynamics simulations showed a highly collapsed, hydrogen-bonded denatured state containing turn and β-sheet structure and few nucleating helices in an otherwise unfolded ensemble. An irregular β-hairpin element that connects helices in the native fold was poised to be formed. The surprising observation of β-structure in regions that form helices in the native state is reconciled by a generic low-energy pathway from the northwest quadrant of Ramachandran space to the helical basin present under folding conditions, proposed recently. Our results show that, indeed, rapid nucleation of helix emanates from β-structure formed early within a collapsed ensemble of unfolded conformers.}, author = {Thukral, Lipi and Schwarze, Simone and Daidone, Isabella and Neuweiler, Hannes}, journal = {Journal of Molecular Biology}, keywords = {hannes}, title = {β-Structure within the Denatured State of the Helical Protein Domain BBL}, year = 2015 }

  %0 Journal Article %1 thukralstructure %A Thukral, Lipi %A Schwarze, Simone %A Daidone, Isabella %A Neuweiler, Hannes %D 2015 %J Journal of Molecular Biology %R http://dx.doi.org/10.1016/j.jmb.2015.08.007 %T β-Structure within the Denatured State of the Helical Protein Domain BBL %U http://www.sciencedirect.com/science/article/pii/S0022283615004520 %X Abstract Protein denatured states are the origin of both healthy and toxic conformational species. Denatured states of ultrafast folding proteins are of interest in mechanistic studies because they are energetically close to the kinetic bottleneck of folding. However, their transient nature makes them elusive to experiment. Here, we generated the denatured state of the helical domain BBL that is poised to fold in microseconds by a single-point mutation and combined circular dichroism spectroscopy, single-molecule fluorescence fluctuation analysis, and computer simulation to characterize its structure and dynamics. Circular dichroism showed a largely unfolded ensemble with marginal helix but significant β-sheet content. Main-chain structure and dynamics were unaffected by side-chain interactions that stabilize the native state, as revealed by site-directed mutagenesis and nanosecond loop closure kinetics probed by fluorescence correlation spectroscopy. Replica-exchange and constant-temperature molecular dynamics simulations showed a highly collapsed, hydrogen-bonded denatured state containing turn and β-sheet structure and few nucleating helices in an otherwise unfolded ensemble. An irregular β-hairpin element that connects helices in the native fold was poised to be formed. The surprising observation of β-structure in regions that form helices in the native state is reconciled by a generic low-energy pathway from the northwest quadrant of Ramachandran space to the helical basin present under folding conditions, proposed recently. Our results show that, indeed, rapid nucleation of helix emanates from β-structure formed early within a collapsed ensemble of unfolded conformers.

• PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching. Sauer, Markus; Neuweiler, Hannes. In Methods Mol Biol - Fluorescence Spectroscopy and Microscopy, Bd. 1076, Y. Engelborghs, A. J. Visser (Hrsg.), S. 597–615. Humana Press, 2014.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples.

  @incollection{year={2014}, abstract = {Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples.}, author = {Sauer, Markus and Neuweiler, Hannes}, booktitle = {Methods Mol Biol - Fluorescence Spectroscopy and Microscopy}, editor = {Engelborghs, Yves and Visser, Antonie J.W.G.}, keywords = {hannes}, pages = {597-615}, publisher = {Humana Press}, series = {Methods in Molecular Biology}, title = {PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching}, volume = 1076, year = 2014 }

  %0 Book Section %1 year={2014} %A Sauer, Markus %A Neuweiler, Hannes %B Methods Mol Biol - Fluorescence Spectroscopy and Microscopy %D 2014 %E Engelborghs, Yves %E Visser, Antonie J.W.G. %I Humana Press %P 597-615 %R 10.1007/978-1-62703-649-8_27 %T PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching %U http://dx.doi.org/10.1007/978-1-62703-649-8_27 %V 1076 %X Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples. %@ 978-1-62703-648-1
• Microsecond folding and domain motions of a spider silk protein structural switch. Ries, Julia; Schwarze, Simone; Johnson, Christopher M.; Neuweiler, Hannes. In Journal of the American Chemical Society, (ja), S. null. 2014.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Web spiders rapidly assemble protein monomers, so-called spidroins, into extraordinarily tough silk fibers. The process involves the pH-triggered self-association of the spidroin N-terminal domain (NTD), which contains a structural switch connecting spidroins to super-molecules. Single-molecule spectroscopy can detect conformational heterogeneity that is hidden to conventional methods, but motions of the NTD are beyond the resolution limit. Here, we engineered probes for 1-nm conformational changes based on the phenomenon of fluorescence quenching by photoinduced electron transfer into the isolated NTD of a spidroin from the nursery web spider Euprosthenops australis. Correlation analysis of single-molecule fluorescence fluctuations uncovered site-dependent nano-to-microsecond movement of secondary and tertiary structure. Kinetic amplitudes were most pronounced for helices that are part of the association interface and where structural studies show large displacements between monomeric and dimeric conformations. A single tryptophan at the center of the five-helix bundle toggled conformations in ~100 µs and in a pH-dependent manner. Equilibrium denaturation and temperature-jump relaxation experiments revealed cooperative and ultrafast folding in only 60 µs. We deduced a free-energy surface that exhibits native-state ruggedness with apparently similar barrier heights to folding and native motions. Observed equilibrium dynamics within the domain suggest a conformational selection mechanism in the rapid association of spidroins through their NTDs during silk synthesis by web spiders.

  @article{doi:10.1021/ja508760a, abstract = {Web spiders rapidly assemble protein monomers, so-called spidroins, into extraordinarily tough silk fibers. The process involves the pH-triggered self-association of the spidroin N-terminal domain (NTD), which contains a structural switch connecting spidroins to super-molecules. Single-molecule spectroscopy can detect conformational heterogeneity that is hidden to conventional methods, but motions of the NTD are beyond the resolution limit. Here, we engineered probes for 1-nm conformational changes based on the phenomenon of fluorescence quenching by photoinduced electron transfer into the isolated NTD of a spidroin from the nursery web spider Euprosthenops australis. Correlation analysis of single-molecule fluorescence fluctuations uncovered site-dependent nano-to-microsecond movement of secondary and tertiary structure. Kinetic amplitudes were most pronounced for helices that are part of the association interface and where structural studies show large displacements between monomeric and dimeric conformations. A single tryptophan at the center of the five-helix bundle toggled conformations in  100 µs and in a pH-dependent manner. Equilibrium denaturation and temperature-jump relaxation experiments revealed cooperative and ultrafast folding in only 60 µs. We deduced a free-energy surface that exhibits native-state ruggedness with apparently similar barrier heights to folding and native motions. Observed equilibrium dynamics within the domain suggest a conformational selection mechanism in the rapid association of spidroins through their NTDs during silk synthesis by web spiders.}, author = {Ries, Julia and Schwarze, Simone and Johnson, Christopher M. and Neuweiler, Hannes}, journal = {Journal of the American Chemical Society}, keywords = {hannes}, note = {PMID: 25382060}, number = {ja}, pages = {null}, title = {Microsecond folding and domain motions of a spider silk protein structural switch}, year = 2014 }

  %0 Journal Article %1 doi:10.1021/ja508760a %A Ries, Julia %A Schwarze, Simone %A Johnson, Christopher M. %A Neuweiler, Hannes %D 2014 %J Journal of the American Chemical Society %N ja %P null %R 10.1021/ja508760a %T Microsecond folding and domain motions of a spider silk protein structural switch %U http://dx.doi.org/10.1021/ja508760a %X Web spiders rapidly assemble protein monomers, so-called spidroins, into extraordinarily tough silk fibers. The process involves the pH-triggered self-association of the spidroin N-terminal domain (NTD), which contains a structural switch connecting spidroins to super-molecules. Single-molecule spectroscopy can detect conformational heterogeneity that is hidden to conventional methods, but motions of the NTD are beyond the resolution limit. Here, we engineered probes for 1-nm conformational changes based on the phenomenon of fluorescence quenching by photoinduced electron transfer into the isolated NTD of a spidroin from the nursery web spider Euprosthenops australis. Correlation analysis of single-molecule fluorescence fluctuations uncovered site-dependent nano-to-microsecond movement of secondary and tertiary structure. Kinetic amplitudes were most pronounced for helices that are part of the association interface and where structural studies show large displacements between monomeric and dimeric conformations. A single tryptophan at the center of the five-helix bundle toggled conformations in ~100 µs and in a pH-dependent manner. Equilibrium denaturation and temperature-jump relaxation experiments revealed cooperative and ultrafast folding in only 60 µs. We deduced a free-energy surface that exhibits native-state ruggedness with apparently similar barrier heights to folding and native motions. Observed equilibrium dynamics within the domain suggest a conformational selection mechanism in the rapid association of spidroins through their NTDs during silk synthesis by web spiders.

• The N-terminal domains of spider silk proteins assemble ultrafast and protected from charge screening. Schwarze, Simone; Zwettler, Fabian U.; Johnson, Christopher M.; Neuweiler, Hannes. In Nat Commun, 4. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2013.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Web spiders assemble spidroin monomers into silk fibres of unrivalled tensile strength at remarkably high spinning speeds of up to 1 m s−1. The spidroin N-terminal domain contains a charge-driven, pH-sensitive relay that controls self-association by an elusive mechanism. The underlying kinetics have not yet been reported. Here we engineer a fluorescence switch into the isolated N-terminal domain from spidroin 1 of the major ampullate gland of the nursery web spider E. australis that monitors dimerization. We observe ultrafast association that is surprisingly insensitive to salt, contrasting the classical screening effects in accelerated, charged protein interfaces. To gain deeper mechanistic insight, we mutate each of the protonatable residue side chains and probe their contributions. Two vicinal aspartic acids are critically involved in an unusual process of accelerated protein association that is protected from screening by electrolytes, potentially facilitating the rapid synthesis of silk fibres by web spiders.

  @article{schwarze2013nterminal, abstract = {Web spiders assemble spidroin monomers into silk fibres of unrivalled tensile strength at remarkably high spinning speeds of up to 1 m s−1. The spidroin N-terminal domain contains a charge-driven, pH-sensitive relay that controls self-association by an elusive mechanism. The underlying kinetics have not yet been reported. Here we engineer a fluorescence switch into the isolated N-terminal domain from spidroin 1 of the major ampullate gland of the nursery web spider E. australis that monitors dimerization. We observe ultrafast association that is surprisingly insensitive to salt, contrasting the classical screening effects in accelerated, charged protein interfaces. To gain deeper mechanistic insight, we mutate each of the protonatable residue side chains and probe their contributions. Two vicinal aspartic acids are critically involved in an unusual process of accelerated protein association that is protected from screening by electrolytes, potentially facilitating the rapid synthesis of silk fibres by web spiders.}, author = {Schwarze, Simone and Zwettler, Fabian U. and Johnson, Christopher M. and Neuweiler, Hannes}, journal = {Nat Commun}, keywords = {hannes}, month = 11, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {The N-terminal domains of spider silk proteins assemble ultrafast and protected from charge screening}, volume = 4, year = 2013 }

  %0 Journal Article %1 schwarze2013nterminal %A Schwarze, Simone %A Zwettler, Fabian U. %A Johnson, Christopher M. %A Neuweiler, Hannes %D 2013 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Commun %T The N-terminal domains of spider silk proteins assemble ultrafast and protected from charge screening %U http://dx.doi.org/10.1038/ncomms3815 %V 4 %X Web spiders assemble spidroin monomers into silk fibres of unrivalled tensile strength at remarkably high spinning speeds of up to 1 m s−1. The spidroin N-terminal domain contains a charge-driven, pH-sensitive relay that controls self-association by an elusive mechanism. The underlying kinetics have not yet been reported. Here we engineer a fluorescence switch into the isolated N-terminal domain from spidroin 1 of the major ampullate gland of the nursery web spider E. australis that monitors dimerization. We observe ultrafast association that is surprisingly insensitive to salt, contrasting the classical screening effects in accelerated, charged protein interfaces. To gain deeper mechanistic insight, we mutate each of the protonatable residue side chains and probe their contributions. Two vicinal aspartic acids are critically involved in an unusual process of accelerated protein association that is protected from screening by electrolytes, potentially facilitating the rapid synthesis of silk fibres by web spiders.

• Long-Range Modulation of Chain Motions within the Intrinsically Disordered Transactivation Domain of Tumor Suppressor p53. Lum, Jenifer K.; Neuweiler, Hannes; Fersht, Alan R. In Journal of the American Chemical Society, 134(3), S. 1617–1622. 2012.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification.

  @article{doi:10.1021/ja2078619, abstract = {The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification.}, author = {Lum, Jenifer K. and Neuweiler, Hannes and Fersht, Alan R.}, journal = {Journal of the American Chemical Society}, keywords = {hannes}, number = 3, pages = {1617-1622}, title = {Long-Range Modulation of Chain Motions within the Intrinsically Disordered Transactivation Domain of Tumor Suppressor p53}, volume = 134, year = 2012 }

  %0 Journal Article %1 doi:10.1021/ja2078619 %A Lum, Jenifer K. %A Neuweiler, Hannes %A Fersht, Alan R. %D 2012 %J Journal of the American Chemical Society %N 3 %P 1617-1622 %R 10.1021/ja2078619 %T Long-Range Modulation of Chain Motions within the Intrinsically Disordered Transactivation Domain of Tumor Suppressor p53 %U http://pubs.acs.org/doi/abs/10.1021/ja2078619 %V 134 %X The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification.

• Intrinsic Motions in the N-Terminal Domain of an Ionotropic Glutamate Receptor Detected by Fluorescence Correlation Spectroscopy. Jensen, Mette H.; Sukumaran, Madhav; Johnson, Christopher M.; Greger, Ingo H.; Neuweiler, Hannes. In Journal of Molecular Biology, 414(1), S. 96–105. 2011.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the central nervous system and play key roles in brain development and disease. iGluRs have two distinct extracellular domains, but the functional role of the distal N-terminal domain (NTD) is poorly understood. Crystal structures of the NTD from some non-N-methyl-d-aspartate (NMDA) iGluRs are consistent with a rigid body that facilitates receptor assembly but suggest an additional dynamic role that could modulate signaling. Here, we moved beyond spatial and temporal limitations of conventional protein single-molecule spectroscopy by employing correlation analysis of extrinsic oxazine fluorescence fluctuations. We observed nanosecond (ns)-to-microsecond (μs) motions of loop segments and helices within a region of an AMPA-type iGluR NTD, which has been identified previously to be structurally variable. Our data reveal that the AMPA receptor NTD undergoes rapid conformational fluctuations, suggesting an inherent allosteric capacity for this domain in addition to its established assembly function.

  @article{Jensen201196, abstract = {Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the central nervous system and play key roles in brain development and disease. iGluRs have two distinct extracellular domains, but the functional role of the distal N-terminal domain (NTD) is poorly understood. Crystal structures of the NTD from some non-N-methyl-d-aspartate (NMDA) iGluRs are consistent with a rigid body that facilitates receptor assembly but suggest an additional dynamic role that could modulate signaling. Here, we moved beyond spatial and temporal limitations of conventional protein single-molecule spectroscopy by employing correlation analysis of extrinsic oxazine fluorescence fluctuations. We observed nanosecond (ns)-to-microsecond (μs) motions of loop segments and helices within a region of an AMPA-type iGluR NTD, which has been identified previously to be structurally variable. Our data reveal that the AMPA receptor NTD undergoes rapid conformational fluctuations, suggesting an inherent allosteric capacity for this domain in addition to its established assembly function.}, author = {Jensen, Mette H. and Sukumaran, Madhav and Johnson, Christopher M. and Greger, Ingo H. and Neuweiler, Hannes}, journal = {Journal of Molecular Biology}, keywords = {hannes}, number = 1, pages = {96 - 105}, title = {Intrinsic Motions in the N-Terminal Domain of an Ionotropic Glutamate Receptor Detected by Fluorescence Correlation Spectroscopy}, volume = 414, year = 2011 }

  %0 Journal Article %1 Jensen201196 %A Jensen, Mette H. %A Sukumaran, Madhav %A Johnson, Christopher M. %A Greger, Ingo H. %A Neuweiler, Hannes %D 2011 %J Journal of Molecular Biology %N 1 %P 96 - 105 %R 10.1016/j.jmb.2011.09.037 %T Intrinsic Motions in the N-Terminal Domain of an Ionotropic Glutamate Receptor Detected by Fluorescence Correlation Spectroscopy %U http://www.sciencedirect.com/science/article/pii/S0022283611010771 %V 414 %X Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the central nervous system and play key roles in brain development and disease. iGluRs have two distinct extracellular domains, but the functional role of the distal N-terminal domain (NTD) is poorly understood. Crystal structures of the NTD from some non-N-methyl-d-aspartate (NMDA) iGluRs are consistent with a rigid body that facilitates receptor assembly but suggest an additional dynamic role that could modulate signaling. Here, we moved beyond spatial and temporal limitations of conventional protein single-molecule spectroscopy by employing correlation analysis of extrinsic oxazine fluorescence fluctuations. We observed nanosecond (ns)-to-microsecond (μs) motions of loop segments and helices within a region of an AMPA-type iGluR NTD, which has been identified previously to be structurally variable. Our data reveal that the AMPA receptor NTD undergoes rapid conformational fluctuations, suggesting an inherent allosteric capacity for this domain in addition to its established assembly function.
• Backbone-Driven Collapse in Unfolded Protein Chains. Teufel, Daniel P.; Johnson, Christopher M.; Lum, Jenifer K.; Neuweiler, Hannes. In Journal of Molecular Biology, 409(2), S. 250–262. 2011.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Collapse of unfolded protein chains is an early event in folding. It affects structural properties of intrinsically disordered proteins, which take a considerable fraction of the human proteome. Collapse is generally believed to be driven by hydrophobic forces imposed by the presence of nonpolar amino acid side chains. Contributions from backbone hydrogen bonds to protein folding and stability, however, are controversial. To date, the experimental dissection of side-chain and backbone contributions has not yet been achieved because both types of interactions are integral parts of protein structure. Here, we realized this goal by applying mutagenesis and chemical modification on a set of disordered peptides and proteins. We measured the protein dimensions and kinetics of intra-chain diffusion of modified polypeptides at the level of individual molecules using fluorescence correlation spectroscopy, thereby avoiding artifacts commonly caused by aggregation of unfolded protein material in bulk. We found no contributions from side chains to collapse but, instead, identified backbone interactions as a source sufficient to form globules of native-like dimensions. The presence of backbone hydrogen bonds decreased polypeptide water solubility dramatically and accelerated the nanosecond kinetics of loop closure, in agreement with recent predictions from computer simulation. The presence of side chains, instead, slowed loop closure and modulated the dimensions of intrinsically disordered domains. It appeared that the transient formation of backbone interactions facilitates the diffusive search for productive conformations at the early stage of folding and within intrinsically disordered proteins.

  @article{Teufel2011250, abstract = {Collapse of unfolded protein chains is an early event in folding. It affects structural properties of intrinsically disordered proteins, which take a considerable fraction of the human proteome. Collapse is generally believed to be driven by hydrophobic forces imposed by the presence of nonpolar amino acid side chains. Contributions from backbone hydrogen bonds to protein folding and stability, however, are controversial. To date, the experimental dissection of side-chain and backbone contributions has not yet been achieved because both types of interactions are integral parts of protein structure. Here, we realized this goal by applying mutagenesis and chemical modification on a set of disordered peptides and proteins. We measured the protein dimensions and kinetics of intra-chain diffusion of modified polypeptides at the level of individual molecules using fluorescence correlation spectroscopy, thereby avoiding artifacts commonly caused by aggregation of unfolded protein material in bulk. We found no contributions from side chains to collapse but, instead, identified backbone interactions as a source sufficient to form globules of native-like dimensions. The presence of backbone hydrogen bonds decreased polypeptide water solubility dramatically and accelerated the nanosecond kinetics of loop closure, in agreement with recent predictions from computer simulation. The presence of side chains, instead, slowed loop closure and modulated the dimensions of intrinsically disordered domains. It appeared that the transient formation of backbone interactions facilitates the diffusive search for productive conformations at the early stage of folding and within intrinsically disordered proteins.}, author = {Teufel, Daniel P. and Johnson, Christopher M. and Lum, Jenifer K. and Neuweiler, Hannes}, journal = {Journal of Molecular Biology}, keywords = {hannes}, number = 2, pages = {250 - 262}, title = {Backbone-Driven Collapse in Unfolded Protein Chains}, volume = 409, year = 2011 }

  %0 Journal Article %1 Teufel2011250 %A Teufel, Daniel P. %A Johnson, Christopher M. %A Lum, Jenifer K. %A Neuweiler, Hannes %D 2011 %J Journal of Molecular Biology %N 2 %P 250 - 262 %R DOI: 10.1016/j.jmb.2011.03.066 %T Backbone-Driven Collapse in Unfolded Protein Chains %U http://www.sciencedirect.com/science/article/pii/S002228361100355X %V 409 %X Collapse of unfolded protein chains is an early event in folding. It affects structural properties of intrinsically disordered proteins, which take a considerable fraction of the human proteome. Collapse is generally believed to be driven by hydrophobic forces imposed by the presence of nonpolar amino acid side chains. Contributions from backbone hydrogen bonds to protein folding and stability, however, are controversial. To date, the experimental dissection of side-chain and backbone contributions has not yet been achieved because both types of interactions are integral parts of protein structure. Here, we realized this goal by applying mutagenesis and chemical modification on a set of disordered peptides and proteins. We measured the protein dimensions and kinetics of intra-chain diffusion of modified polypeptides at the level of individual molecules using fluorescence correlation spectroscopy, thereby avoiding artifacts commonly caused by aggregation of unfolded protein material in bulk. We found no contributions from side chains to collapse but, instead, identified backbone interactions as a source sufficient to form globules of native-like dimensions. The presence of backbone hydrogen bonds decreased polypeptide water solubility dramatically and accelerated the nanosecond kinetics of loop closure, in agreement with recent predictions from computer simulation. The presence of side chains, instead, slowed loop closure and modulated the dimensions of intrinsically disordered domains. It appeared that the transient formation of backbone interactions facilitates the diffusive search for productive conformations at the early stage of folding and within intrinsically disordered proteins.

• Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains. Daidone, Isabella; Neuweiler, Hannes; Doose, Sören; Sauer, Markus; Smith, Jeremy C. In PLOS COMPUTATIONAL BIOLOGY, 6(1). PUBLIC LIBRARY SCIENCE, 185 BERRY ST, STE 1300, SAN FRANCISCO, CA 94107 USA, 2010.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20-100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient beta-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events.

  @article{Daidone2010, abstract = {Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20-100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient beta-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events.}, address = {185 BERRY ST, STE 1300, SAN FRANCISCO, CA 94107 USA}, author = {Daidone, Isabella and Neuweiler, Hannes and Doose, Sören and Sauer, Markus and Smith, Jeremy C.}, journal = {PLOS COMPUTATIONAL BIOLOGY}, keywords = {hannes}, month = {01}, number = 1, publisher = {PUBLIC LIBRARY SCIENCE}, title = {Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains}, type = {Article}, volume = 6, year = 2010 }

  %0 Journal Article %1 Daidone2010 %A Daidone, Isabella %A Neuweiler, Hannes %A Doose, Sören %A Sauer, Markus %A Smith, Jeremy C. %C 185 BERRY ST, STE 1300, SAN FRANCISCO, CA 94107 USA %D 2010 %I PUBLIC LIBRARY SCIENCE %J PLOS COMPUTATIONAL BIOLOGY %N 1 %T Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains %U http://dx.doi.org/10.1371/journal.pcbi.1000645 %V 6 %X Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20-100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient beta-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events.
• Carboxyl pK(a) Values and Acid Denaturation of BBL. Arbely, Eyal; Rutherford, Trevor J.; Neuweiler, Hannes; Sharpe, Timothy D.; Ferguson, Neil; Fersht, Alan R. In JOURNAL OF MOLECULAR BIOLOGY, 403(2), S. 313–327. ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND, 2010.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK(a) values of all the carboxylic residues in this pH range. We employed C-13 direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pKa values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pKa values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pKa, and, using thermodynamic cycles, we could calculate their pK(a)s in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured C-13(alpha) chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state. (C) 2010 Elsevier Ltd. All rights reserved.

  @article{Arbely2010, abstract = {The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK(a) values of all the carboxylic residues in this pH range. We employed C-13 direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pKa values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pKa values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pKa, and, using thermodynamic cycles, we could calculate their pK(a)s in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured C-13(alpha) chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state. (C) 2010 Elsevier Ltd. All rights reserved.}, address = {24-28 OVAL RD, LONDON NW1 7DX, ENGLAND}, author = {Arbely, Eyal and Rutherford, Trevor J. and Neuweiler, Hannes and Sharpe, Timothy D. and Ferguson, Neil and Fersht, Alan R.}, journal = {JOURNAL OF MOLECULAR BIOLOGY}, keywords = {hannes}, month = 10, number = 2, pages = {313-327}, publisher = {ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD}, title = {Carboxyl pK(a) Values and Acid Denaturation of BBL}, type = {Article}, volume = 403, year = 2010 }

  %0 Journal Article %1 Arbely2010 %A Arbely, Eyal %A Rutherford, Trevor J. %A Neuweiler, Hannes %A Sharpe, Timothy D. %A Ferguson, Neil %A Fersht, Alan R. %C 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND %D 2010 %I ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD %J JOURNAL OF MOLECULAR BIOLOGY %N 2 %P 313-327 %T Carboxyl pK(a) Values and Acid Denaturation of BBL %U http://dx.doi.org/10.1016/j.jmb.2010.08.052 %V 403 %X The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK(a) values of all the carboxylic residues in this pH range. We employed C-13 direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pKa values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pKa values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pKa, and, using thermodynamic cycles, we could calculate their pK(a)s in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured C-13(alpha) chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state. (C) 2010 Elsevier Ltd. All rights reserved.
• The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces. Arbely, Eyal; Neuweiler, Hannes; Sharpe, Timothy D.; Johnson, Christopher M.; Fersht, Alan R. In PROTEIN SCIENCE, 19(9), S. 1704–1713. JOHN WILEY & SONS INC, 111 RIVER ST, HOBOKEN, NJ 07030 USA, 2010.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Huckel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution.

  @article{Arbely2010a, abstract = {Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Huckel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution.}, address = {111 RIVER ST, HOBOKEN, NJ 07030 USA}, author = {Arbely, Eyal and Neuweiler, Hannes and Sharpe, Timothy D. and Johnson, Christopher M. and Fersht, Alan R.}, journal = {PROTEIN SCIENCE}, keywords = {hannes}, month = {09}, number = 9, pages = {1704-1713}, publisher = {JOHN WILEY \& SONS INC}, title = {The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces}, type = {Article}, volume = 19, year = 2010 }

  %0 Journal Article %1 Arbely2010a %A Arbely, Eyal %A Neuweiler, Hannes %A Sharpe, Timothy D. %A Johnson, Christopher M. %A Fersht, Alan R. %C 111 RIVER ST, HOBOKEN, NJ 07030 USA %D 2010 %I JOHN WILEY & SONS INC %J PROTEIN SCIENCE %N 9 %P 1704-1713 %T The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces %U http://dx.doi.org/10.1002/pro.453 %V 19 %X Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Huckel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution.
• Kinetics of chain motions within a protein-folding intermediate. Neuweiler, Hannes; Banachewicz, Wiktor; Fersht, Alan R. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 107(51), S. 22106–22110. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2010.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Small proteins can fold remarkably rapidly, even in mu s. What limits their rate of folding? The Engrailed homeodomain is a particularly well-characterized example, which folds ultrafast via an intermediate, I, of solved structure. It is a puzzle that the helix2-turn-helix3 motif of the 3-helix bundle forms in approximately 2 mu s, but the final docking of preformed helix1 in I requires approximately 20 mu s. Simulation and structural data suggest that nonnative interactions may slow down helix docking. Here we report the direct measurement of chain motions in I by using photoinduced electron transfer fluorescence-quenching correlation spectroscopy (PET-FCS). We use a mutant that traps I at physiological ionic strength but refolds at higher ionic strength. A single Trp in helix3 quenches the fluorescence of an extrinsic label on contact with it. We placed the label along the sequence to probe segmental chain motions. At high ionic strength, we found two relaxations for all probed positions on the 2- and 20-mu s time scale, corresponding to the known folding processes, and a 200-ns phase attributable to loop closure kinetics in the unfolded state. At low ionic strength, we found only the 2-mu s and 200-ns phase for labels in the helix2-turn-helix3 motif of I, because the native state is not significantly populated. But for labels in helix1 we observed an additional approximately 10-mu s phase showing that it was moving slowly, with a rate constant similar to that for overall folding under native conditions. Folding was rate-limited by chain motions on a rough energy surface where nonnative interactions constrain motion.

  @article{Neuweiler2010, abstract = {Small proteins can fold remarkably rapidly, even in mu s. What limits their rate of folding? The Engrailed homeodomain is a particularly well-characterized example, which folds ultrafast via an intermediate, I, of solved structure. It is a puzzle that the helix2-turn-helix3 motif of the 3-helix bundle forms in approximately 2 mu s, but the final docking of preformed helix1 in I requires approximately 20 mu s. Simulation and structural data suggest that nonnative interactions may slow down helix docking. Here we report the direct measurement of chain motions in I by using photoinduced electron transfer fluorescence-quenching correlation spectroscopy (PET-FCS). We use a mutant that traps I at physiological ionic strength but refolds at higher ionic strength. A single Trp in helix3 quenches the fluorescence of an extrinsic label on contact with it. We placed the label along the sequence to probe segmental chain motions. At high ionic strength, we found two relaxations for all probed positions on the 2- and 20-mu s time scale, corresponding to the known folding processes, and a 200-ns phase attributable to loop closure kinetics in the unfolded state. At low ionic strength, we found only the 2-mu s and 200-ns phase for labels in the helix2-turn-helix3 motif of I, because the native state is not significantly populated. But for labels in helix1 we observed an additional approximately 10-mu s phase showing that it was moving slowly, with a rate constant similar to that for overall folding under native conditions. Folding was rate-limited by chain motions on a rough energy surface where nonnative interactions constrain motion.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Neuweiler, Hannes and Banachewicz, Wiktor and Fersht, Alan R.}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {hannes}, month = 12, number = 51, pages = {22106-22110}, publisher = {NATL ACAD SCIENCES}, title = {Kinetics of chain motions within a protein-folding intermediate}, type = {Article}, volume = 107, year = 2010 }

  %0 Journal Article %1 Neuweiler2010 %A Neuweiler, Hannes %A Banachewicz, Wiktor %A Fersht, Alan R. %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2010 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 51 %P 22106-22110 %T Kinetics of chain motions within a protein-folding intermediate %U http://dx.doi.org/10.1073/pnas.1011666107 %V 107 %X Small proteins can fold remarkably rapidly, even in mu s. What limits their rate of folding? The Engrailed homeodomain is a particularly well-characterized example, which folds ultrafast via an intermediate, I, of solved structure. It is a puzzle that the helix2-turn-helix3 motif of the 3-helix bundle forms in approximately 2 mu s, but the final docking of preformed helix1 in I requires approximately 20 mu s. Simulation and structural data suggest that nonnative interactions may slow down helix docking. Here we report the direct measurement of chain motions in I by using photoinduced electron transfer fluorescence-quenching correlation spectroscopy (PET-FCS). We use a mutant that traps I at physiological ionic strength but refolds at higher ionic strength. A single Trp in helix3 quenches the fluorescence of an extrinsic label on contact with it. We placed the label along the sequence to probe segmental chain motions. At high ionic strength, we found two relaxations for all probed positions on the 2- and 20-mu s time scale, corresponding to the known folding processes, and a 200-ns phase attributable to loop closure kinetics in the unfolded state. At low ionic strength, we found only the 2-mu s and 200-ns phase for labels in the helix2-turn-helix3 motif of I, because the native state is not significantly populated. But for labels in helix1 we observed an additional approximately 10-mu s phase showing that it was moving slowly, with a rate constant similar to that for overall folding under native conditions. Folding was rate-limited by chain motions on a rough energy surface where nonnative interactions constrain motion.

• Reply to Campos et al.: Direct observation versus ambiguous kinetics and thermodynamics. Huang, Fang; Ying, Liming; Neuweiler, Hannes; Fersht, Alan R. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 106(52), S. E140. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2009.
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  @article{Huang2009, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Huang, Fang and Ying, Liming and Neuweiler, Hannes and Fersht, Alan R.}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {hannes}, month = 12, number = 52, pages = {E140}, publisher = {NATL ACAD SCIENCES}, title = {Reply to Campos et al.: Direct observation versus ambiguous kinetics and thermodynamics}, type = {Letter}, volume = 106, year = 2009 }

  %0 Journal Article %1 Huang2009 %A Huang, Fang %A Ying, Liming %A Neuweiler, Hannes %A Fersht, Alan R. %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2009 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 52 %P E140 %T Reply to Campos et al.: Direct observation versus ambiguous kinetics and thermodynamics %U http://dx.doi.org/10.1073/pnas.0913321107 %V 106
• Direct observation of ultrafast folding and denatured state dynamics in single protein molecules. Neuweiler, Hannes; Johnson, Christopher M.; Fersht, Alan R. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 106(44), S. 18569–18574. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2009.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than approximate to 100 mu s and distances greater than approximate to 2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-mu s decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type ({''}D-phys''). D-phys had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the ``speed limit'' of folding. D-phys exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.

  @article{Neuweiler2009, abstract = {Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than approximate to 100 mu s and distances greater than approximate to 2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-mu s decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type ({''}D-phys''). D-phys had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the ``speed limit'' of folding. D-phys exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Neuweiler, Hannes and Johnson, Christopher M. and Fersht, Alan R.}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {hannes}, month = 11, number = 44, pages = {18569-18574}, publisher = {NATL ACAD SCIENCES}, title = {Direct observation of ultrafast folding and denatured state dynamics in single protein molecules}, type = {Article}, volume = 106, year = 2009 }

  %0 Journal Article %1 Neuweiler2009 %A Neuweiler, Hannes %A Johnson, Christopher M. %A Fersht, Alan R. %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2009 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 44 %P 18569-18574 %T Direct observation of ultrafast folding and denatured state dynamics in single protein molecules %U http://dx.doi.org/10.1073/pnas.0910860106 %V 106 %X Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than approximate to 100 mu s and distances greater than approximate to 2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-mu s decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type ({''}D-phys''). D-phys had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the ``speed limit'' of folding. D-phys exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.
• Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules. Doose, Sören; Neuweiler, Hannes; Sauer, Markus. In CHEMPHYSCHEM, 10(9-10, Sp. Iss. SI), S. 1389–1398. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2009.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties, for example, the amino acid tryptophan or the nucleobase guanine, can quench fluorescence upon van der Waals contact and thus report-on molecular contact. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and oligonucleotides. Whereas dynamic quenching transiently influences quantum yield and fluorescence life-time of the fluorophore, static quenching in pi-stacked complexes efficiently suppresses fluorescence emission over time scales longer than the fluorescence lifetime. Static quenching therefore provides sufficient contrast to be observed at the single-molecule level. Here, we review complex formation and static quenching of different fluorophores by various molecular compounds, discuss applications as reporter system for macromolecular dynamics, and give illustrating examples.

  @article{Doose2009, abstract = {Photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties, for example, the amino acid tryptophan or the nucleobase guanine, can quench fluorescence upon van der Waals contact and thus report-on molecular contact. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and oligonucleotides. Whereas dynamic quenching transiently influences quantum yield and fluorescence life-time of the fluorophore, static quenching in pi-stacked complexes efficiently suppresses fluorescence emission over time scales longer than the fluorescence lifetime. Static quenching therefore provides sufficient contrast to be observed at the single-molecule level. Here, we review complex formation and static quenching of different fluorophores by various molecular compounds, discuss applications as reporter system for macromolecular dynamics, and give illustrating examples.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Doose, Sören and Neuweiler, Hannes and Sauer, Markus}, journal = {CHEMPHYSCHEM}, keywords = {hannes}, month = {07}, number = {9-10, Sp. Iss. SI}, pages = {1389-1398}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules}, type = {Review}, volume = 10, year = 2009 }

  %0 Journal Article %1 Doose2009 %A Doose, Sören %A Neuweiler, Hannes %A Sauer, Markus %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2009 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 9-10, Sp. Iss. SI %P 1389-1398 %T Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules %U http://dx.doi.org/10.1002/cphc.200900238 %V 10 %X Photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties, for example, the amino acid tryptophan or the nucleobase guanine, can quench fluorescence upon van der Waals contact and thus report-on molecular contact. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and oligonucleotides. Whereas dynamic quenching transiently influences quantum yield and fluorescence life-time of the fluorophore, static quenching in pi-stacked complexes efficiently suppresses fluorescence emission over time scales longer than the fluorescence lifetime. Static quenching therefore provides sufficient contrast to be observed at the single-molecule level. Here, we review complex formation and static quenching of different fluorophores by various molecular compounds, discuss applications as reporter system for macromolecular dynamics, and give illustrating examples.
• The Folding Mechanism of BBL: Plasticity of Transition-State Structure Observed within an Ultrafast Folding Protein Family. Neuweiler, Hannes; Sharpe, Timothy D.; Rutherford, Trevor J.; Johnson, Christopher M.; Allen, Mark D.; Ferguson, Neil; Fersht, Alan R. In JOURNAL OF MOLECULAR BIOLOGY, 390(5), S. 1060–1073. ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD, 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND, 2009.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Studies on members of protein families with similar structures but divergent sequences provide insights into the effects of sequence composition on the mechanism of folding. Members of the peripheral subunit-binding domain (PSBD) family fold ultrafast and approach the smallest size for cooperatively folding proteins. Phi-Value analysis of the PSBDs E3BD and POB reveals folding via nucleation-condensation through structurally very similar, polarized transition states. Here, we present a Phi-value analysis of the family member BBL and found that it also folds by a nucleation-condensation mechanism. The mean Phi values of BBL, E3BD, and POB were near identical, indicating similar fractions of non-covalent interactions being formed in the transition state. Despite the overall conservation of folding mechanism in this protein family, however, the pattern of Phi values determined for BBL revealed a larger dispersion of the folding nucleus across the entire structure, and the transition state was less polarized. The observed plasticity of transition-state structure can be rationalized by the different helix-forming propensities of PSBD sequences. The very strong helix propensity in the first helix of BBL, relative to E3BD and POB, appears to recruit more structure formation in that helix in the transition state at the expense of weaker interactions in the second helix. Differences in sequence composition can modulate transition-state structure of even the smallest natural protein domains. (C) 2009 Elsevier Ltd. All rights reserved.

  @article{Neuweiler2009b, abstract = {Studies on members of protein families with similar structures but divergent sequences provide insights into the effects of sequence composition on the mechanism of folding. Members of the peripheral subunit-binding domain (PSBD) family fold ultrafast and approach the smallest size for cooperatively folding proteins. Phi-Value analysis of the PSBDs E3BD and POB reveals folding via nucleation-condensation through structurally very similar, polarized transition states. Here, we present a Phi-value analysis of the family member BBL and found that it also folds by a nucleation-condensation mechanism. The mean Phi values of BBL, E3BD, and POB were near identical, indicating similar fractions of non-covalent interactions being formed in the transition state. Despite the overall conservation of folding mechanism in this protein family, however, the pattern of Phi values determined for BBL revealed a larger dispersion of the folding nucleus across the entire structure, and the transition state was less polarized. The observed plasticity of transition-state structure can be rationalized by the different helix-forming propensities of PSBD sequences. The very strong helix propensity in the first helix of BBL, relative to E3BD and POB, appears to recruit more structure formation in that helix in the transition state at the expense of weaker interactions in the second helix. Differences in sequence composition can modulate transition-state structure of even the smallest natural protein domains. (C) 2009 Elsevier Ltd. All rights reserved.}, address = {24-28 OVAL RD, LONDON NW1 7DX, ENGLAND}, author = {Neuweiler, Hannes and Sharpe, Timothy D. and Rutherford, Trevor J. and Johnson, Christopher M. and Allen, Mark D. and Ferguson, Neil and Fersht, Alan R.}, journal = {JOURNAL OF MOLECULAR BIOLOGY}, keywords = {hannes}, month = {07}, number = 5, pages = {1060-1073}, publisher = {ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD}, title = {The Folding Mechanism of BBL: Plasticity of Transition-State Structure Observed within an Ultrafast Folding Protein Family}, type = {Article}, volume = 390, year = 2009 }

  %0 Journal Article %1 Neuweiler2009b %A Neuweiler, Hannes %A Sharpe, Timothy D. %A Rutherford, Trevor J. %A Johnson, Christopher M. %A Allen, Mark D. %A Ferguson, Neil %A Fersht, Alan R. %C 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND %D 2009 %I ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD %J JOURNAL OF MOLECULAR BIOLOGY %N 5 %P 1060-1073 %T The Folding Mechanism of BBL: Plasticity of Transition-State Structure Observed within an Ultrafast Folding Protein Family %U http://dx.doi.org/10.1016/j.jmb.2009.05.011 %V 390 %X Studies on members of protein families with similar structures but divergent sequences provide insights into the effects of sequence composition on the mechanism of folding. Members of the peripheral subunit-binding domain (PSBD) family fold ultrafast and approach the smallest size for cooperatively folding proteins. Phi-Value analysis of the PSBDs E3BD and POB reveals folding via nucleation-condensation through structurally very similar, polarized transition states. Here, we present a Phi-value analysis of the family member BBL and found that it also folds by a nucleation-condensation mechanism. The mean Phi values of BBL, E3BD, and POB were near identical, indicating similar fractions of non-covalent interactions being formed in the transition state. Despite the overall conservation of folding mechanism in this protein family, however, the pattern of Phi values determined for BBL revealed a larger dispersion of the folding nucleus across the entire structure, and the transition state was less polarized. The observed plasticity of transition-state structure can be rationalized by the different helix-forming propensities of PSBD sequences. The very strong helix propensity in the first helix of BBL, relative to E3BD and POB, appears to recruit more structure formation in that helix in the transition state at the expense of weaker interactions in the second helix. Differences in sequence composition can modulate transition-state structure of even the smallest natural protein domains. (C) 2009 Elsevier Ltd. All rights reserved.
• Downhill versus Barrier-Limited Folding of BBL 2: Mechanistic Insights from Kinetics of Folding Monitored by Independent Tryptophan Probes. Neuweiler, Hannes; Sharpe, Timothy D.; Johnson, Christopher M.; Teufel, Daniel P.; Ferguson, Neil; Fersht, Alan R. In JOURNAL OF MOLECULAR BIOLOGY, 387(4), S. 975–985. ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD, 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND, 2009.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Barrier-free downhill folding has been proposed for the peripheral subunit-binding domain BBL. To date, ultrafast kinetic experiments on BBL, which are crucial for a mechanistic understanding of folding, have been hampered by the lack of good intrinsic spectroscopic probes. Here, we present a detailed kinetic characterization of three single-point tryptophan mutants of BBL that have suitable fluorescence properties for following microsecond and nanosecond folding kinetics using temperature jump fluorescence spectroscopy. Experiments were performed at pH 7, which is optimal for stability and minimizes complications that arise from the presence of ail alternative native-state conformation of BBL at lower pH. We examined the dependence of rate and equilibrium constants on concentration of denaturant and found that they follow well-established laws allowing kinetic transients to be related to events in folding and compared with equilibrium data. Logarithms of rate constants versus denaturant concentration yielded plots (chevrons) that are characteristic of barrier-limited folding for all mutants investigated, including a truncated sequence that was previously used in the proposal of downhill folding. The thermodynamic quantities calculated from the rate constants were in excellent agreement with those directly determined from equilibrium denaturation based on empirical two-state equations. We found that sequence truncation of BBL as used in studies proposing downhill folding leads to a large loss in helical content and protein stability, which were exacerbated at the low pH used in those studies. The kinetics and equilibria of folding of BBL fit to conventional barrier-limited kinetics. (C) 2009 Elsevier Ltd. All rights reserved.

  @article{Neuweiler2009a, abstract = {Barrier-free downhill folding has been proposed for the peripheral subunit-binding domain BBL. To date, ultrafast kinetic experiments on BBL, which are crucial for a mechanistic understanding of folding, have been hampered by the lack of good intrinsic spectroscopic probes. Here, we present a detailed kinetic characterization of three single-point tryptophan mutants of BBL that have suitable fluorescence properties for following microsecond and nanosecond folding kinetics using temperature jump fluorescence spectroscopy. Experiments were performed at pH 7, which is optimal for stability and minimizes complications that arise from the presence of ail alternative native-state conformation of BBL at lower pH. We examined the dependence of rate and equilibrium constants on concentration of denaturant and found that they follow well-established laws allowing kinetic transients to be related to events in folding and compared with equilibrium data. Logarithms of rate constants versus denaturant concentration yielded plots (chevrons) that are characteristic of barrier-limited folding for all mutants investigated, including a truncated sequence that was previously used in the proposal of downhill folding. The thermodynamic quantities calculated from the rate constants were in excellent agreement with those directly determined from equilibrium denaturation based on empirical two-state equations. We found that sequence truncation of BBL as used in studies proposing downhill folding leads to a large loss in helical content and protein stability, which were exacerbated at the low pH used in those studies. The kinetics and equilibria of folding of BBL fit to conventional barrier-limited kinetics. (C) 2009 Elsevier Ltd. All rights reserved.}, address = {24-28 OVAL RD, LONDON NW1 7DX, ENGLAND}, author = {Neuweiler, Hannes and Sharpe, Timothy D. and Johnson, Christopher M. and Teufel, Daniel P. and Ferguson, Neil and Fersht, Alan R.}, journal = {JOURNAL OF MOLECULAR BIOLOGY}, keywords = {hannes}, month = {04}, number = 4, pages = {975-985}, publisher = {ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD}, title = {Downhill versus Barrier-Limited Folding of BBL 2: Mechanistic Insights from Kinetics of Folding Monitored by Independent Tryptophan Probes}, type = {Article}, volume = 387, year = 2009 }

  %0 Journal Article %1 Neuweiler2009a %A Neuweiler, Hannes %A Sharpe, Timothy D. %A Johnson, Christopher M. %A Teufel, Daniel P. %A Ferguson, Neil %A Fersht, Alan R. %C 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND %D 2009 %I ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD %J JOURNAL OF MOLECULAR BIOLOGY %N 4 %P 975-985 %T Downhill versus Barrier-Limited Folding of BBL 2: Mechanistic Insights from Kinetics of Folding Monitored by Independent Tryptophan Probes %U http://dx.doi.org/10.1016/j.jmb.2008.12.056 %V 387 %X Barrier-free downhill folding has been proposed for the peripheral subunit-binding domain BBL. To date, ultrafast kinetic experiments on BBL, which are crucial for a mechanistic understanding of folding, have been hampered by the lack of good intrinsic spectroscopic probes. Here, we present a detailed kinetic characterization of three single-point tryptophan mutants of BBL that have suitable fluorescence properties for following microsecond and nanosecond folding kinetics using temperature jump fluorescence spectroscopy. Experiments were performed at pH 7, which is optimal for stability and minimizes complications that arise from the presence of ail alternative native-state conformation of BBL at lower pH. We examined the dependence of rate and equilibrium constants on concentration of denaturant and found that they follow well-established laws allowing kinetic transients to be related to events in folding and compared with equilibrium data. Logarithms of rate constants versus denaturant concentration yielded plots (chevrons) that are characteristic of barrier-limited folding for all mutants investigated, including a truncated sequence that was previously used in the proposal of downhill folding. The thermodynamic quantities calculated from the rate constants were in excellent agreement with those directly determined from equilibrium denaturation based on empirical two-state equations. We found that sequence truncation of BBL as used in studies proposing downhill folding leads to a large loss in helical content and protein stability, which were exacerbated at the low pH used in those studies. The kinetics and equilibria of folding of BBL fit to conventional barrier-limited kinetics. (C) 2009 Elsevier Ltd. All rights reserved.

• Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy. Schuettpelz, Mark; Schoening, Jan C.; Doose, Sören; Neuweiler, Hannes; Peters, Elisabeth; Staiger, Dorothee; Sauer, Markus. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 130(29), S. 9507–9513. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2008.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.

  @article{Schuettpelz2008, abstract = {The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Schuettpelz, Mark and Schoening, Jan C. and Doose, Sören and Neuweiler, Hannes and Peters, Elisabeth and Staiger, Dorothee and Sauer, Markus}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {hannes}, month = {07}, number = 29, pages = {9507-9513}, publisher = {AMER CHEMICAL SOC}, title = {Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy}, type = {Article}, volume = 130, year = 2008 }

  %0 Journal Article %1 Schuettpelz2008 %A Schuettpelz, Mark %A Schoening, Jan C. %A Doose, Sören %A Neuweiler, Hannes %A Peters, Elisabeth %A Staiger, Dorothee %A Sauer, Markus %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2008 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 29 %P 9507-9513 %T Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy %U http://dx.doi.org/10.1021/ja801994z %V 130 %X The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.

• A highly sensitive particle agglutination assay for the detection of P53 autoantibodies in patients with lung cancer. Agaylan, Ashraf; Binder, Daniel; Sauer, Markus; Neuweiler, Hannes; Meyer, Oliver; Kiesewetter, Holger; Salama, Abdulgabar. In Cancer, 110(11), S. 2502–2506. Wiley Subscription Services, Inc., A Wiley Company, 2007.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Abstract BACKGROUND.Numerous assays have been described for the detection of p53 autoantibodies. These assays are highly specific with low sensitivity. In this report, the authors describe a highly sensitive and simple particle agglutination immunoassay using superparamagnetic particles for capturing p5 autoantibodies, p53 protein, and p53 protein-antibody complexes from large volumes of serum samples (2 mL).METHODS.Superparamagnetic particles were coated with different peptides spanning the entire p53 protein. These particles were incubated with serum samples from healthy blood donors (n = 180), from patients without malignancies (n = 27), and from patients with various forms of lung cancer (n = 166). The particles were washed and placed into the reaction chamber of a gel card. After centrifugation, agglutination results were read visually. Positive reactions were defined by a layer of particles on top of the gel or agglutinated particles dispersed through the gel matrix.RESULTS.Depending on the peptide used, p53 autoantibodies were detected in from 17.5% to 35% of the investigated patients with lung cancer. By using a commercially available enzyme-linked immunoadsorbent assay (ELISA) kit, p53 autoantibodies were detected in only 3% of those patients. P53 protein and p53 protein-antibody complexes were not detected in patients with lung cancer (n = 20).CONCLUSIONS.The newly developed assay was easy to perform and had sensitivity superior to that of the currently available p53 ELISAs. Cancer 2007. © 2007 American Cancer Society.

  @article{CNCR:CNCR23057, abstract = {Abstract BACKGROUND.Numerous assays have been described for the detection of p53 autoantibodies. These assays are highly specific with low sensitivity. In this report, the authors describe a highly sensitive and simple particle agglutination immunoassay using superparamagnetic particles for capturing p5 autoantibodies, p53 protein, and p53 protein-antibody complexes from large volumes of serum samples (2 mL).METHODS.Superparamagnetic particles were coated with different peptides spanning the entire p53 protein. These particles were incubated with serum samples from healthy blood donors (n = 180), from patients without malignancies (n = 27), and from patients with various forms of lung cancer (n = 166). The particles were washed and placed into the reaction chamber of a gel card. After centrifugation, agglutination results were read visually. Positive reactions were defined by a layer of particles on top of the gel or agglutinated particles dispersed through the gel matrix.RESULTS.Depending on the peptide used, p53 autoantibodies were detected in from 17.5% to 35% of the investigated patients with lung cancer. By using a commercially available enzyme-linked immunoadsorbent assay (ELISA) kit, p53 autoantibodies were detected in only 3% of those patients. P53 protein and p53 protein-antibody complexes were not detected in patients with lung cancer (n = 20).CONCLUSIONS.The newly developed assay was easy to perform and had sensitivity superior to that of the currently available p53 ELISAs. Cancer 2007. © 2007 American Cancer Society.}, author = {Agaylan, Ashraf and Binder, Daniel and Sauer, Markus and Neuweiler, Hannes and Meyer, Oliver and Kiesewetter, Holger and Salama, Abdulgabar}, journal = {Cancer}, keywords = {hannes}, number = 11, pages = {2502–2506}, publisher = {Wiley Subscription Services, Inc., A Wiley Company}, title = {A highly sensitive particle agglutination assay for the detection of P53 autoantibodies in patients with lung cancer}, volume = 110, year = 2007 }

  %0 Journal Article %1 CNCR:CNCR23057 %A Agaylan, Ashraf %A Binder, Daniel %A Sauer, Markus %A Neuweiler, Hannes %A Meyer, Oliver %A Kiesewetter, Holger %A Salama, Abdulgabar %D 2007 %I Wiley Subscription Services, Inc., A Wiley Company %J Cancer %N 11 %P 2502--2506 %R 10.1002/cncr.23057 %T A highly sensitive particle agglutination assay for the detection of P53 autoantibodies in patients with lung cancer %U http://dx.doi.org/10.1002/cncr.23057 %V 110 %X Abstract BACKGROUND.Numerous assays have been described for the detection of p53 autoantibodies. These assays are highly specific with low sensitivity. In this report, the authors describe a highly sensitive and simple particle agglutination immunoassay using superparamagnetic particles for capturing p5 autoantibodies, p53 protein, and p53 protein-antibody complexes from large volumes of serum samples (2 mL).METHODS.Superparamagnetic particles were coated with different peptides spanning the entire p53 protein. These particles were incubated with serum samples from healthy blood donors (n = 180), from patients without malignancies (n = 27), and from patients with various forms of lung cancer (n = 166). The particles were washed and placed into the reaction chamber of a gel card. After centrifugation, agglutination results were read visually. Positive reactions were defined by a layer of particles on top of the gel or agglutinated particles dispersed through the gel matrix.RESULTS.Depending on the peptide used, p53 autoantibodies were detected in from 17.5% to 35% of the investigated patients with lung cancer. By using a commercially available enzyme-linked immunoadsorbent assay (ELISA) kit, p53 autoantibodies were detected in only 3% of those patients. P53 protein and p53 protein-antibody complexes were not detected in patients with lung cancer (n = 20).CONCLUSIONS.The newly developed assay was easy to perform and had sensitivity superior to that of the currently available p53 ELISAs. Cancer 2007. © 2007 American Cancer Society.
• Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy. Neuweiler, Hannes; Lollmann, Marc; Doose, Sören; Sauer, Markus. In JOURNAL OF MOLECULAR BIOLOGY, 365(3), S. 856–869. ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD, 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND, 2007.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides, have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides. consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in ``off{''} and ``on{''} switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypepticle chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in k unfolded proteins can still proceed quite efficiently in spite of hindered L diffusion caused by high macromolecular content. (c) 2006 Elsevier Ltd. All rights reserved.

  @article{Neuweiler2007, abstract = {Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides, have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides. consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in ``off{''} and ``on{''} switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypepticle chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in k unfolded proteins can still proceed quite efficiently in spite of hindered L diffusion caused by high macromolecular content. (c) 2006 Elsevier Ltd. All rights reserved.}, address = {24-28 OVAL RD, LONDON NW1 7DX, ENGLAND}, author = {Neuweiler, Hannes and Lollmann, Marc and Doose, Sören and Sauer, Markus}, journal = {JOURNAL OF MOLECULAR BIOLOGY}, keywords = {hannes}, month = {01}, number = 3, pages = {856-869}, publisher = {ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD}, title = {Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy}, type = {Article}, volume = 365, year = 2007 }

  %0 Journal Article %1 Neuweiler2007 %A Neuweiler, Hannes %A Lollmann, Marc %A Doose, Sören %A Sauer, Markus %C 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND %D 2007 %I ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD %J JOURNAL OF MOLECULAR BIOLOGY %N 3 %P 856-869 %T Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy %U http://dx.doi.org/10.1016/j.jmb.2006.10.021 %V 365 %X Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides, have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides. consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in ``off{''} and ``on{''} switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypepticle chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in k unfolded proteins can still proceed quite efficiently in spite of hindered L diffusion caused by high macromolecular content. (c) 2006 Elsevier Ltd. All rights reserved.
• Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix. Doose, Sören; Neuweiler, Hannes; Barsch, Hannes; Sauer, Markus. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 104(44), S. 17400–17405. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2007.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Forster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics Of Poly-L-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.

  @article{Doose2007c, abstract = {Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Forster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics Of Poly-L-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Doose, Sören and Neuweiler, Hannes and Barsch, Hannes and Sauer, Markus}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {hannes}, month = 10, number = 44, pages = {17400-17405}, publisher = {NATL ACAD SCIENCES}, title = {Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix}, type = {Article}, volume = 104, year = 2007 }

  %0 Journal Article %1 Doose2007c %A Doose, Sören %A Neuweiler, Hannes %A Barsch, Hannes %A Sauer, Markus %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2007 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 44 %P 17400-17405 %T Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix %U http://dx.doi.org/10.1073/pnas.0705605104 %V 104 %X Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Forster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics Of Poly-L-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.

• UV Fluorescence Lifetime Imaging Microscopy: A Label-Free Method for Detection and Quantification of Protein Interactions. Schüttpelz, Mark; Müller, Christian; Neuweiler, Hannes; Sauer, Markus. In Analytical Chemistry, 78(3), S. 663–669. 2006.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 ± 0.6) × 109 M-1, represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays

  @article{doi:10.1021/ac051938j, abstract = {Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 ± 0.6) × 109 M-1, represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays}, author = {Schüttpelz, Mark and Müller, Christian and Neuweiler, Hannes and Sauer, Markus}, journal = {Analytical Chemistry}, keywords = {hannes}, note = {PMID: 16448037}, number = 3, pages = {663-669}, title = {UV Fluorescence Lifetime Imaging Microscopy: A Label-Free Method for Detection and Quantification of Protein Interactions}, volume = 78, year = 2006 }

  %0 Journal Article %1 doi:10.1021/ac051938j %A Schüttpelz, Mark %A Müller, Christian %A Neuweiler, Hannes %A Sauer, Markus %D 2006 %J Analytical Chemistry %N 3 %P 663-669 %R 10.1021/ac051938j %T UV Fluorescence Lifetime Imaging Microscopy: A Label-Free Method for Detection and Quantification of Protein Interactions %U http://pubs.acs.org/doi/abs/10.1021/ac051938j %V 78 %X Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 ± 0.6) × 109 M-1, represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays
• The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy. Kim, J; Doose, S; Neuweiler, H; Sauer, M. In NUCLEIC ACIDS RESEARCH, 34(9), S. 2516–2527. OXFORD UNIV PRESS, GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND, 2006.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC-dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC-dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC-dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.

  @article{Kim2006, abstract = {Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC-dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC-dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC-dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.}, address = {GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND}, author = {Kim, J and Doose, S and Neuweiler, H and Sauer, M}, journal = {NUCLEIC ACIDS RESEARCH}, keywords = {hannes}, number = 9, pages = {2516-2527}, publisher = {OXFORD UNIV PRESS}, title = {The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy}, type = {Article}, volume = 34, year = 2006 }

  %0 Journal Article %1 Kim2006 %A Kim, J %A Doose, S %A Neuweiler, H %A Sauer, M %C GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND %D 2006 %I OXFORD UNIV PRESS %J NUCLEIC ACIDS RESEARCH %N 9 %P 2516-2527 %T The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy %U http://dx.doi.org/10.1093/nar/gkl221 %V 34 %X Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC-dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC-dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC-dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.

• A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate. Neuweiler, H; Doose, S; Sauer, M. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 102(46), S. 16650–16655. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2005.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  The role of polypeptide collapse and formation of intermediates in protein folding is still under debate. Miniproteins, small globular peptide structures, serve as ideal model systems to study the basic principles that govern folding. Experimental investigations of folding dynamics of such small systems, however, turn out to be challenging, because requirements for high temporal and spatial resolution have to be met simultaneously. Here, we demonstrate how selective quenching of an extrinsic fluorescent label by the amino acid tryptophan (Trp) can be used to probe folding dynamics of Trp-cage (TC), the smallest protein known to date. Using fluorescence correlation spectroscopy, we monitor folding transitions as well as conformational flexibility in the denatured state of the 20-residue protein under thermodynamic equilibrium conditions with nanosecond time resolution. Besides microsecond folding kinetics, we reveal hierarchical folding of TC, hidden to previous experimental studies. We show that specific collapse of the peptide to a molten globule-like intermediate enhances folding efficiency considerably. A single point mutation destabilizes the intermediate, switching the protein to two-state folding behavior and slowing down the folding process. Our results underscore the importance of preformed structure in the denatured state for folding of even the smallest globular structures. A unique method emerges for monitoring conformational dynamics and ultrafast folding events of polypeptides at the nanometer scale.

  @article{Neuweiler2005, abstract = {The role of polypeptide collapse and formation of intermediates in protein folding is still under debate. Miniproteins, small globular peptide structures, serve as ideal model systems to study the basic principles that govern folding. Experimental investigations of folding dynamics of such small systems, however, turn out to be challenging, because requirements for high temporal and spatial resolution have to be met simultaneously. Here, we demonstrate how selective quenching of an extrinsic fluorescent label by the amino acid tryptophan (Trp) can be used to probe folding dynamics of Trp-cage (TC), the smallest protein known to date. Using fluorescence correlation spectroscopy, we monitor folding transitions as well as conformational flexibility in the denatured state of the 20-residue protein under thermodynamic equilibrium conditions with nanosecond time resolution. Besides microsecond folding kinetics, we reveal hierarchical folding of TC, hidden to previous experimental studies. We show that specific collapse of the peptide to a molten globule-like intermediate enhances folding efficiency considerably. A single point mutation destabilizes the intermediate, switching the protein to two-state folding behavior and slowing down the folding process. Our results underscore the importance of preformed structure in the denatured state for folding of even the smallest globular structures. A unique method emerges for monitoring conformational dynamics and ultrafast folding events of polypeptides at the nanometer scale.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Neuweiler, H and Doose, S and Sauer, M}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {hannes}, month = 11, number = 46, pages = {16650-16655}, publisher = {NATL ACAD SCIENCES}, title = {A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate}, type = {Article}, volume = 102, year = 2005 }

  %0 Journal Article %1 Neuweiler2005 %A Neuweiler, H %A Doose, S %A Sauer, M %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2005 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 46 %P 16650-16655 %T A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate %U http://dx.doi.org/10.1073/pnas.0507351102 %V 102 %X The role of polypeptide collapse and formation of intermediates in protein folding is still under debate. Miniproteins, small globular peptide structures, serve as ideal model systems to study the basic principles that govern folding. Experimental investigations of folding dynamics of such small systems, however, turn out to be challenging, because requirements for high temporal and spatial resolution have to be met simultaneously. Here, we demonstrate how selective quenching of an extrinsic fluorescent label by the amino acid tryptophan (Trp) can be used to probe folding dynamics of Trp-cage (TC), the smallest protein known to date. Using fluorescence correlation spectroscopy, we monitor folding transitions as well as conformational flexibility in the denatured state of the 20-residue protein under thermodynamic equilibrium conditions with nanosecond time resolution. Besides microsecond folding kinetics, we reveal hierarchical folding of TC, hidden to previous experimental studies. We show that specific collapse of the peptide to a molten globule-like intermediate enhances folding efficiency considerably. A single point mutation destabilizes the intermediate, switching the protein to two-state folding behavior and slowing down the folding process. Our results underscore the importance of preformed structure in the denatured state for folding of even the smallest globular structures. A unique method emerges for monitoring conformational dynamics and ultrafast folding events of polypeptides at the nanometer scale.
• A close look at fluorescence quenching of organic dyes by tryptophan. Doose, S; Neuweiler, H; Sauer, M. In CHEMPHYSCHEM, 6(11), S. 2277–2285. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2005.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M-1, 2) static quenching with a bimolecular association constant K-s of 61 M-1, and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M-1. The latter two are characterized as nonfluorescent complexes, formed with approximate to 30% efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 2030 kJmol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.

  @article{Doose2005a, abstract = {Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M-1, 2) static quenching with a bimolecular association constant K-s of 61 M-1, and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M-1. The latter two are characterized as nonfluorescent complexes, formed with approximate to 30\% efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 2030 kJmol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Doose, S and Neuweiler, H and Sauer, M}, journal = {CHEMPHYSCHEM}, keywords = {hannes}, month = 11, number = 11, pages = {2277-2285}, publisher = {WILEY-V C H VERLAG GMBH}, title = {A close look at fluorescence quenching of organic dyes by tryptophan}, type = {Article}, volume = 6, year = 2005 }

  %0 Journal Article %1 Doose2005a %A Doose, S %A Neuweiler, H %A Sauer, M %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2005 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 11 %P 2277-2285 %T A close look at fluorescence quenching of organic dyes by tryptophan %U http://dx.doi.org/10.1002/cphc.200500191 %V 6 %X Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M-1, 2) static quenching with a bimolecular association constant K-s of 61 M-1, and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M-1. The latter two are characterized as nonfluorescent complexes, formed with approximate to 30% efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 2030 kJmol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.
• Exploring life by single-molecule fluorescence spectroscopy. Neuweiler, H; Sauer, M. In ANALYTICAL CHEMISTRY, 77(9), S. 178A-185A. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2005.
  • [ BibTeX ]
  • [ EndNote ]
  @article{Neuweiler2005a, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Neuweiler, H and Sauer, M}, journal = {ANALYTICAL CHEMISTRY}, keywords = {hannes}, month = {05}, number = 9, pages = {178A-185A}, publisher = {AMER CHEMICAL SOC}, title = {Exploring life by single-molecule fluorescence spectroscopy}, type = {Article}, volume = 77, year = 2005 }

  %0 Journal Article %1 Neuweiler2005a %A Neuweiler, H %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2005 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 9 %P 178A-185A %T Exploring life by single-molecule fluorescence spectroscopy %V 77
• Monitoring antibody binding events in homogeneous solution by single-molecule fluorescence spectroscopy. Scheffler, S; Sauer, M; Neuweiler, H. In ZEITSCHRIFT FUR PHYSIKALISCHE CHEMIE-INTERNATIONAL JOURNAL OF RESEARCH IN PHYSICAL CHEMISTRY & CHEMICAL PHYSICS, 219(5), S. 665–678. R OLDENBOURG VERLAG, LEKTORAT MINT, POSTFACH 80 13 60, D-81613 MUNICH, GERMANY, 2005.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  We demonstrate the potential of modern confocal fluorescence microscopy in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing simultaneously two parameters: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Furthermore, we demonstrate that the strong fluorescence increase upon binding can also be used to directly detect p53 autoantibodies from human blood serum samples in fluorescence intensity time traces. Our data demonstrate that new refined single-molecule fluorescence techniques in combination with quenched peptide epitopes open new possibilities for the reliable detection of antibody binding events in homogeneous solution.

  @article{Scheffler2005, abstract = {We demonstrate the potential of modern confocal fluorescence microscopy in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing simultaneously two parameters: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Furthermore, we demonstrate that the strong fluorescence increase upon binding can also be used to directly detect p53 autoantibodies from human blood serum samples in fluorescence intensity time traces. Our data demonstrate that new refined single-molecule fluorescence techniques in combination with quenched peptide epitopes open new possibilities for the reliable detection of antibody binding events in homogeneous solution.}, address = {LEKTORAT MINT, POSTFACH 80 13 60, D-81613 MUNICH, GERMANY}, author = {Scheffler, S and Sauer, M and Neuweiler, H}, journal = {ZEITSCHRIFT FUR PHYSIKALISCHE CHEMIE-INTERNATIONAL JOURNAL OF RESEARCH IN PHYSICAL CHEMISTRY \& CHEMICAL PHYSICS}, keywords = {hannes}, number = 5, pages = {665-678}, publisher = {R OLDENBOURG VERLAG}, title = {Monitoring antibody binding events in homogeneous solution by single-molecule fluorescence spectroscopy}, type = {Article}, volume = 219, year = 2005 }

  %0 Journal Article %1 Scheffler2005 %A Scheffler, S %A Sauer, M %A Neuweiler, H %C LEKTORAT MINT, POSTFACH 80 13 60, D-81613 MUNICH, GERMANY %D 2005 %I R OLDENBOURG VERLAG %J ZEITSCHRIFT FUR PHYSIKALISCHE CHEMIE-INTERNATIONAL JOURNAL OF RESEARCH IN PHYSICAL CHEMISTRY & CHEMICAL PHYSICS %N 5 %P 665-678 %T Monitoring antibody binding events in homogeneous solution by single-molecule fluorescence spectroscopy %U http://dx.doi.org/10.1524/zpch.219.5.665.64323 %V 219 %X We demonstrate the potential of modern confocal fluorescence microscopy in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing simultaneously two parameters: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Furthermore, we demonstrate that the strong fluorescence increase upon binding can also be used to directly detect p53 autoantibodies from human blood serum samples in fluorescence intensity time traces. Our data demonstrate that new refined single-molecule fluorescence techniques in combination with quenched peptide epitopes open new possibilities for the reliable detection of antibody binding events in homogeneous solution.

• Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level. Neuweiler, H; Sauer, M. In CURRENT PHARMACEUTICAL BIOTECHNOLOGY, 5(3), S. 285–298. BENTHAM SCIENCE PUBL LTD, EXECUTIVE STE Y26, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATES, 2004.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  This mini-review describes how single-molecule sensitive fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in biopolymers. Single-pair FRET experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angstroms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.

  @article{Neuweiler2004, abstract = {This mini-review describes how single-molecule sensitive fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in biopolymers. Single-pair FRET experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angstroms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.}, address = {EXECUTIVE STE Y26, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATES}, author = {Neuweiler, H and Sauer, M}, journal = {CURRENT PHARMACEUTICAL BIOTECHNOLOGY}, keywords = {hannes}, month = {06}, number = 3, pages = {285-298}, publisher = {BENTHAM SCIENCE PUBL LTD}, title = {Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level}, type = {Review}, volume = 5, year = 2004 }

  %0 Journal Article %1 Neuweiler2004 %A Neuweiler, H %A Sauer, M %C EXECUTIVE STE Y26, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATES %D 2004 %I BENTHAM SCIENCE PUBL LTD %J CURRENT PHARMACEUTICAL BIOTECHNOLOGY %N 3 %P 285-298 %T Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level %U http://dx.doi.org/10.2174/1389201043376896 %V 5 %X This mini-review describes how single-molecule sensitive fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in biopolymers. Single-pair FRET experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angstroms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.

• Fluorescence quenching of dyes by tryptophan: Interactions at atomic detail from combination of experiment and computer simulation. Vaiana, AC; Neuweiler, H; Schulz, A; Wolfrum, J; Sauer, M; Smith, JC. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 125(47), S. 14564–14572. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Fluorescence spectroscopy and molecular dynamics (MD) simulation are combined to characterize the interaction of two organic fluorescent dyes, rhodamine 6G (R6G) and an oxazine derivative (MR121), with the amino acid tryptophan in aqueous solution. Steady-state and time-resolved fluorescence quenching experiments reveal the formation of essentially nonfluorescent ground-state dye/Trp complexes. The MD simulations are used to elucidate the molecular interaction geometries involved. The MD-derived probability distribution of the distance r between the centers of geometry of the dye and quencher ring systems, P(r), extends to higher distances for R6G than for MR121 due to population in the R6G/Trp system of fluorescent interaction geometries between Trp and the phenyl ring and ester group of the dye. The consequence of this is the experimental finding that under the conditions used in the simulations about 25% of the R6G dye is fluorescent in comparison with 10% of the MR121. Combining the above findings allows determination of the ``quenching distance{''}, r{*}, above which no quenching occurs. r{*} is found to be very similar (similar to5.5 Angstrom) for both dye/Trp systems, corresponding to close to van der Waals contact. Both experimental dynamic Stern-Volmer analysis and the MD trajectories demonstrate that the main determinant of the fluorescence intensity is static quenching. The approach presented is likely to be useful in the structural interpretation of data obtained from fluorescent conjugates commonly used for monitoring the binding and dynamics of biomolecular systems.

  @article{Vaiana2003a, abstract = {Fluorescence spectroscopy and molecular dynamics (MD) simulation are combined to characterize the interaction of two organic fluorescent dyes, rhodamine 6G (R6G) and an oxazine derivative (MR121), with the amino acid tryptophan in aqueous solution. Steady-state and time-resolved fluorescence quenching experiments reveal the formation of essentially nonfluorescent ground-state dye/Trp complexes. The MD simulations are used to elucidate the molecular interaction geometries involved. The MD-derived probability distribution of the distance r between the centers of geometry of the dye and quencher ring systems, P(r), extends to higher distances for R6G than for MR121 due to population in the R6G/Trp system of fluorescent interaction geometries between Trp and the phenyl ring and ester group of the dye. The consequence of this is the experimental finding that under the conditions used in the simulations about 25\% of the R6G dye is fluorescent in comparison with 10\% of the MR121. Combining the above findings allows determination of the ``quenching distance{''}, r{*}, above which no quenching occurs. r{*} is found to be very similar (similar to5.5 Angstrom) for both dye/Trp systems, corresponding to close to van der Waals contact. Both experimental dynamic Stern-Volmer analysis and the MD trajectories demonstrate that the main determinant of the fluorescence intensity is static quenching. The approach presented is likely to be useful in the structural interpretation of data obtained from fluorescent conjugates commonly used for monitoring the binding and dynamics of biomolecular systems.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Vaiana, AC and Neuweiler, H and Schulz, A and Wolfrum, J and Sauer, M and Smith, JC}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {hannes}, month = 11, number = 47, pages = {14564-14572}, publisher = {AMER CHEMICAL SOC}, title = {Fluorescence quenching of dyes by tryptophan: Interactions at atomic detail from combination of experiment and computer simulation}, type = {Article}, volume = 125, year = 2003 }

  %0 Journal Article %1 Vaiana2003a %A Vaiana, AC %A Neuweiler, H %A Schulz, A %A Wolfrum, J %A Sauer, M %A Smith, JC %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 47 %P 14564-14572 %T Fluorescence quenching of dyes by tryptophan: Interactions at atomic detail from combination of experiment and computer simulation %U http://dx.doi.org/10.1021/ja036082j %V 125 %X Fluorescence spectroscopy and molecular dynamics (MD) simulation are combined to characterize the interaction of two organic fluorescent dyes, rhodamine 6G (R6G) and an oxazine derivative (MR121), with the amino acid tryptophan in aqueous solution. Steady-state and time-resolved fluorescence quenching experiments reveal the formation of essentially nonfluorescent ground-state dye/Trp complexes. The MD simulations are used to elucidate the molecular interaction geometries involved. The MD-derived probability distribution of the distance r between the centers of geometry of the dye and quencher ring systems, P(r), extends to higher distances for R6G than for MR121 due to population in the R6G/Trp system of fluorescent interaction geometries between Trp and the phenyl ring and ester group of the dye. The consequence of this is the experimental finding that under the conditions used in the simulations about 25% of the R6G dye is fluorescent in comparison with 10% of the MR121. Combining the above findings allows determination of the ``quenching distance{''}, r{*}, above which no quenching occurs. r{*} is found to be very similar (similar to5.5 Angstrom) for both dye/Trp systems, corresponding to close to van der Waals contact. Both experimental dynamic Stern-Volmer analysis and the MD trajectories demonstrate that the main determinant of the fluorescence intensity is static quenching. The approach presented is likely to be useful in the structural interpretation of data obtained from fluorescent conjugates commonly used for monitoring the binding and dynamics of biomolecular systems.
• Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level. Neuweiler, H; Schulz, A; Bohmer, M; Enderlein, J; Sauer, M. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 125(18), S. 5324–5330. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.

  @article{Neuweiler2003, abstract = {We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Neuweiler, H and Schulz, A and Bohmer, M and Enderlein, J and Sauer, M}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {hannes}, month = {05}, number = 18, pages = {5324-5330}, publisher = {AMER CHEMICAL SOC}, title = {Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level}, type = {Article}, volume = 125, year = 2003 }

  %0 Journal Article %1 Neuweiler2003 %A Neuweiler, H %A Schulz, A %A Bohmer, M %A Enderlein, J %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 18 %P 5324-5330 %T Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level %U http://dx.doi.org/10.1021/ja034040p %V 125 %X We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.

• Detection of Individual p53-Autoantibodies by Using Quenched Peptide-Based Molecular Probes. Neuweiler, Hannes; Schulz, Andreas; Vaiana, Andrea C.; Smith, Jeremy C.; Kaul, Sepp; Wolfrum, Jürgen; Sauer, Markus. In Angewandte Chemie International Edition, 41(24), S. 4769–4773. WILEY-VCH Verlag, 2002.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Single-molecule spectroscopy for cancer diagnostics: Short dye-labeled peptide epitopes derived from human p53 show strong fluorescence quenching owing to efficient electron transfer from a tryptophan residue to the fluorophore (1, see picture). The specific recognition of the peptide epitope by the antibody induces a change in the conformation which is accompanied by a strong increase in fluorescence intensity. The fluorescing antibody–peptide complex can be detected at the single-molecule level even in slightly diluted serum samples by using confocal fluorescence microscopy.

  @article{ANIE:ANIE200290044, abstract = {Single-molecule spectroscopy for cancer diagnostics: Short dye-labeled peptide epitopes derived from human p53 show strong fluorescence quenching owing to efficient electron transfer from a tryptophan residue to the fluorophore (1, see picture). The specific recognition of the peptide epitope by the antibody induces a change in the conformation which is accompanied by a strong increase in fluorescence intensity. The fluorescing antibody–peptide complex can be detected at the single-molecule level even in slightly diluted serum samples by using confocal fluorescence microscopy.}, author = {Neuweiler, Hannes and Schulz, Andreas and Vaiana, Andrea C. and Smith, Jeremy C. and Kaul, Sepp and Wolfrum, Jürgen and Sauer, Markus}, journal = {Angewandte Chemie International Edition}, keywords = {hannes}, number = 24, pages = {4769–4773}, publisher = {WILEY-VCH Verlag}, title = {Detection of Individual p53-Autoantibodies by Using Quenched Peptide-Based Molecular Probes}, volume = 41, year = 2002 }

  %0 Journal Article %1 ANIE:ANIE200290044 %A Neuweiler, Hannes %A Schulz, Andreas %A Vaiana, Andrea C. %A Smith, Jeremy C. %A Kaul, Sepp %A Wolfrum, Jürgen %A Sauer, Markus %D 2002 %I WILEY-VCH Verlag %J Angewandte Chemie International Edition %N 24 %P 4769--4773 %R 10.1002/anie.200290044 %T Detection of Individual p53-Autoantibodies by Using Quenched Peptide-Based Molecular Probes %U http://dx.doi.org/10.1002/anie.200290044 %V 41 %X Single-molecule spectroscopy for cancer diagnostics: Short dye-labeled peptide epitopes derived from human p53 show strong fluorescence quenching owing to efficient electron transfer from a tryptophan residue to the fluorophore (1, see picture). The specific recognition of the peptide epitope by the antibody induces a change in the conformation which is accompanied by a strong increase in fluorescence intensity. The fluorescing antibody–peptide complex can be detected at the single-molecule level even in slightly diluted serum samples by using confocal fluorescence microscopy.