Maik Jacob - Academia.edu (original) (raw)
Papers by Maik Jacob
Journal of Biomolecular Nmr, 2003
For a detailed NMR study of the dynamics of the cold shock protein CspB from Bacillus subtilis, w... more For a detailed NMR study of the dynamics of the cold shock protein CspB from Bacillus subtilis, we determined (15)N transverse and longitudinal relaxation rates and heteronuclear nuclear Overhauser effects at different solvent viscosities. Up to a relative viscosity of 2, which is equivalent to 27% ethylene glycol (EG), the overall correlation time follows the linear Stokes-Einstein equation. At a relative viscosity of 6 (70% EG) the correlation time deviates from linearity by 30%, indicating that CspB tumbles at a higher rate as expected from the solvent viscosity probably due to a preferential binding of water molecules at the protein surface. The corresponding hydrodynamic radii, determined by NMR diffusion experiments, show no variation with viscosity. The amplitudes of intramolecular motions on a sub-nanosecond time scale revealed by an extended Lipari-Szabo analysis were mainly independent of the solvent viscosity. The lower limit of the NMR 'observation window' for the internal correlation time shifts above 0.5 ns at 70% EG, which is directly reflected in the experimentally derived internal correlation times. Chemical exchange contributions to the transverse relaxation rates derived from the Lipari-Szabo approach coincide with the experimentally determined values from the transverse (1)H-(15)N dipolar/(15)N chemical shift anisotropy relaxation interference. These contributions originate from fast protein folding reactions on a millisecond timescale, which get retarded at increased solvent viscosities.
Proceedings of the National Academy of Sciences of the United States of America, May 1, 1997
The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds ... more The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds extremely fast in a reversible N leftrightharpoons U two-state reaction. Both unfolding and refolding are strongly decelerated when the viscosity of the solvent is increased by adding ethylene glycol or sucrose. The folding of CspB thus seems to follow Kramers' model for reactions in which the reactants must diffuse together. It indicates that the compaction of the protein chain occurs in the rate-limiting step of folding. Chain diffusion to a productively collapsed form and the crossing of a high energy barrier are thus tightly coupled in this folding reaction, and the measured reaction rate depends on both the diffusion of the protein chain in the solvent and the magnitude of the activation energy. We suggest that in protein folding an energetic barrier is essential to separate the native from the unfolded conformations of a protein. This barrier protects the ordered structure of a native protein against continuous unfolding by diffusive chain motions and leads to apparent two-state behavior.
Journal of biomolecular NMR, 2003
For a detailed NMR study of the dynamics of the cold shock protein CspB from Bacillus subtilis, w... more For a detailed NMR study of the dynamics of the cold shock protein CspB from Bacillus subtilis, we determined (15)N transverse and longitudinal relaxation rates and heteronuclear nuclear Overhauser effects at different solvent viscosities. Up to a relative viscosity of 2, which is equivalent to 27% ethylene glycol (EG), the overall correlation time follows the linear Stokes-Einstein equation. At a relative viscosity of 6 (70% EG) the correlation time deviates from linearity by 30%, indicating that CspB tumbles at a higher rate as expected from the solvent viscosity probably due to a preferential binding of water molecules at the protein surface. The corresponding hydrodynamic radii, determined by NMR diffusion experiments, show no variation with viscosity. The amplitudes of intramolecular motions on a sub-nanosecond time scale revealed by an extended Lipari-Szabo analysis were mainly independent of the solvent viscosity. The lower limit of the NMR 'observation window' for th...
Nature structural biology, 1999
There has been some debate as to whether protein folding involves diffusive chain motions and thu... more There has been some debate as to whether protein folding involves diffusive chain motions and thus depends on solvent viscosity. The interpretation of folding kinetics in viscous solvents has remained difficult and controversial, in that viscogenic agents affect folding rates not only by increasing solvent viscosity but also by increasing protein stability. By carefully choosing experimental conditions, we can now eliminate the effect on stability and show that the folding dynamics of the cold shock protein CspB are viscosity dependent. Thus Kramers' theory of reaction rates rather than transition state theory should be used to describe this folding reaction.
Schweitzer-Stenner/Peptide Folding, Misfolding, and Nonfold, 2012
The Journal of Physical Chemistry B, 2013
The structural and dynamic properties of a flexible peptidic chain codetermine its biological act... more The structural and dynamic properties of a flexible peptidic chain codetermine its biological activity. These properties are imprinted in intrachain site-to-site distances as well as in diffusion coefficients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of Forster resonance energy transfer (FRET) between two chain sites labeled with a FRET donor and acceptor. Both could be obtained from time-resolved FRET measurements if their individual contributions to the FRET efficiency could be systematically varied. Because the FRET diffusion enhancement (FDE) depends on the donor-fluorescence lifetime, it has been proposed that the FDE can be reduced by shortening the donor lifetime through an external quencher. Benefiting from the high diffusion sensitivity of short-distance FRET, we tested this concept experimentally on a (Gly−Ser) 6 segment labeled with the donor/acceptor pair naphthylalanine/2,3-diazabicyclo[2.2.2]oct-2-ene (NAla/Dbo). Surprisingly, the very effective quencher potassium iodide (KI) had no effect at all on the average donor−acceptor distance, although the donor lifetime was shortened from ca. 36 ns in the absence of KI to ca. 3 ns in the presence of 30 mM KI. We show that the proposed approach had to fail because it is not the experimentally observed but the radiative donor lifetime that controls the FDE. Because of that, any FRET ensemble measurement can easily underestimate diffusion and might be misleading even if it employs the Haas−Steinberg diffusion equation (HSE). An extension of traditional FRET analysis allowed us to evaluate HSE simulations and to corroborate as well as generalize the experimental results. We demonstrate that diffusion-enhanced FRET depends on the radiative donor lifetime as it depends on the diffusion coefficient, a useful symmetry that can directly be applied to distinguish dynamic and structural effects of viscous cosolvents on the polymer chain. We demonstrate that the effective FRET rate and the recovered donor−acceptor distance depend on the quantum yield, most strongly in the absence of diffusion, which has to be accounted for in the interpretation of distance trends monitored by FRET. . Selected donor−acceptor and donor-only labeled peptides composed of Gly−Ser units. Donors are naphthyl-1-L-alanine (NAla) and 5fluoro-L-tryptophan (FTrp); the acceptor is Dbo, whose optically active group is the azo group of the bicyclic chromophore.
Proceedings of the National Academy of Sciences, 1997
The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds ... more The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds extremely fast in a reversible N ª U two-state reaction. Both unfolding and refolding are strongly decelerated when the viscosity of the solvent is increased by adding ethylene glycol or sucrose.
Journal of Molecular Biology, 2009
Most globular protein chains, when transferred from high to low denaturant concentrations, collap... more Most globular protein chains, when transferred from high to low denaturant concentrations, collapse instantly before they refold to their native state. The initial compaction of the protein molecule is assumed to have a key effect on the folding pathway, but it is not known whether the earliest structures formed during or instantly after collapse are defined by local or by non-local interactions-that is, by secondary structural elements or by loop closure of long segments of the protein chain. Stable closure of one or several long loops can reduce the chain entropy at a very early stage and can prevent the protein from following non-productive pathways whose number grows exponentially with the length of the protein chain. In Escherichia coli adenylate kinase (AK), about seven long loops define the topology of the native structure. We selected four loop-forming sections of the chain and probed the time course of loop formation during refolding of AK. We labeled the termini of the loop segments with tryptophan and cysteine-5-amidosalicylic acid. This donor-acceptor pair of probes used with fluorescence resonance excitation energy transfer spectroscopy (FRET) is suitable for detecting very short distances and thus is able to distinguish between random and specific compactions. Refolding of AK was initiated by stopped-flow mixing, followed simultaneously by donor and acceptor fluorescence, and analyzed in terms of energy transfer efficiency and distance. In the collapsed state of AK, observed after the 5-ms dead time of the instrument, one of the selected segments shows a native-like separation of its termini; it forms a loop already in the collapsed state. A second segment that includes the first but is longer by 15 residues shows an almost native-like separation of its termini. In contrast, a segment that is shorter but part of the second segment shows a distance separation of its termini as high as a segment that spans almost the whole protein chain. We conclude that a specific network of non-local interactions, the closure of one or several loops, can play an important role in determining the protein folding pathway at its early phases. . † T. Orevi and E. B. Ishai contributed equally to this work. Abbreviations used: A-AK, AK labeled with a FRET acceptor probe; D-AK, AK labeled with an engineered Trp residue as a FRET donor probe; DA-AK, AK labeled with both donor and acceptor; ETE, energy transfer efficiency; FRET, fluorescence resonance excitation energy transfer; GndHCl, guanidinium hydrochloride; SA, 5-amidosalicylic acid; tr-FRET, time-resolved FRET; wt-AK, Escherichia coli C77S (wild type) adenylate kinase.
Journal of Molecular Biology, 2002
The cold-shock protein CspB folds rapidly in a N Y U two-state reaction via a transition state th... more The cold-shock protein CspB folds rapidly in a N Y U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive ðDV ‡ NU ¼ 16ð^4Þ ml=molÞ and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well ðDV ‡ UN ¼ 53ð^6Þ ml=molÞ and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, DV ‡ UN decreases significantly, suggesting that compact unfolded forms become populated under these conditions. Abbreviations used: CspB, cold-shock protein from Bacillus subtilis; GdmSCN, guanidinium thiocyanate; l, measured rate of a reaction; k ij , microscopic rate constant; DV ‡ app , apparent activation volume; DV ‡ ij , microscopic activation volume; n, n ij , denaturant-dependence of reaction and activation volumes.
Biophysical Chemistry, 2002
The folding reactions of several proteins are well described as diffusional barrier crossing proc... more The folding reactions of several proteins are well described as diffusional barrier crossing processes, which suggests that they should be analyzed by Kramers' rate theory rather than by transition state theory. For the cold shock protein Bc-Csp from Bacillus caldolyticus, we measured stability and folding kinetics, as well as solvent viscosity as a function of temperature and denaturant concentration. Our analysis indicates that diffusional folding reactions can be treated by transition state theory, provided that the temperature and denaturant dependence of the solvent viscosity is properly accounted for, either at the level of the measured rate constants or of the calculated activation parameters. After viscosity correction the activation barriers for folding become less enthalpic and more entropic. The transition from an enthalpic to an entropic folding barrier with increasing temperature is, however, apparent in the data before and after this correction. It is a consequence of the negative activation heat capacity of refolding, which is independent of solvent viscosity. Bc-Csp and its mesophilic homolog Bs-CspB from Bacillus subtilis differ strongly in stability but show identical enthalpic and entropic barriers to refolding. The increased stability of Bc-Csp originates from additional enthalpic interactions that are established after passage through the activated state. As a consequence, the activation enthalpy of unfolding is increased relative to Bs-CspB. ᮊ
Biochemistry, 1999
A protein chain must move relative to the solvent molecules and explore many conformations when i... more A protein chain must move relative to the solvent molecules and explore many conformations when it folds from the extended unfolded state to the compact native state. Experimental and theoretical approaches suggest that diffusional processes in fact contribute to the kinetics of protein folding. We describe here how variations of the solvent viscosity can be employed to uncover the diffusional contributions to a folding reaction and assess the use of transition state theory and Kramers' rate theory for the analysis of protein folding reactions.
Biochemistry, 1999
A pressure-jump apparatus was employed in investigating the kinetics of protein unfolding and ref... more A pressure-jump apparatus was employed in investigating the kinetics of protein unfolding and refolding. In the reaction cell, the pressure can be increased or decreased by 100-160 bar within 50-100 microseconds and then held constant. Thus, unfolding and refolding reactions in the time range from 70 microseconds to 70 s can be followed with this technique. Measurements are possible in the transition regions of thermally or denaturant-induced folding in a wide range of temperatures and solvent conditions. We used this pressure-jump method to determine the temperature dependence of the rate constants of unfolding and refolding of the cold shock protein of Bacillus subtilis and of three variants thereof with Phe --> Ala substitutions in the central beta-sheet region. For all variants, the change in heat capacity occurred in refolding between the unfolded and activated states, suggesting that the overall native-like character of the activated state of folding was not changed by the deletion of individual Phe side chains. The Phe27Ala mutation affected the rate of unfolding only; the Phe15Ala and Phe17Ala mutations changed the kinetics of both unfolding and refolding. Although the activated state of folding of the cold shock protein is overall native-like, individual side chains are still in a non-native environment.
Biochemistry, 2005
A variety of biophysical methods used to study proteins requires protein modification using conju... more A variety of biophysical methods used to study proteins requires protein modification using conjugated molecular probes. Cysteine is the main residue that can be modified without the risk of altering other residues in the protein chain. It is possible to label several cysteines in a protein using highly selective labeling reactions, if the cysteines react at very different rates. The reactivity of a cysteine residue introduced into an exposed surface site depends on the fraction of cysteine in the deprotonated state. Here, it is shown that cysteine reactivity differences can be effectively predicted by an electrostatic model that yields site-specifically the fractions of cysteinate. The model accounts for electrostatic interactions between the cysteinyl anion and side chains, the local protein backbone, and water. The energies of interaction with side chains and the main chain are calculated by using the two different dielectric constants, 40 and 22, respectively. Twenty-six mutants of Escherichia coli adenylate kinase were produced, each containing a single cysteine at the protein surface, and the rates of the reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) were measured. Cysteine residues were chosen on the basis of locations that were expected to allow modification of the protein with minimal risk of perturbing its structure. The reaction rates spanned a range of 6 orders of magnitude. The correlation between predicted fractions of cysteinate and measured reaction rates was strong (R = 92%) and especially high (R = 97%) for cysteines at the helix termini. The approach developed here allows reasonably fast, automated screening of protein surfaces to identify sites that permit efficient preparations of double- or triple-labeled protein.
Angewandte Chemie, 2014
ABSTRACT Die Effektivität von Wirkstoffen und Biomolekülen basiert auf ihrer Fähigkeit, die Lipid... more ABSTRACT Die Effektivität von Wirkstoffen und Biomolekülen basiert auf ihrer Fähigkeit, die Lipidmembran zu passieren. Die Entwicklung von Methoden, diese Transportprozesse direkt und mit hoher Empfindlichkeit zu verfolgen, bleibt eine Herausforderung. Der Einschluss eines chemosensorischen Ensembles, bestehend aus einem makrocyclischen Wirt (p-Sulfonatocalix[4]aren oder Cucurbit[7]uril) und einem Fluoreszenzfarbstoff (Lucigenin oder Berberin) in Liposomen ermöglicht es, den Membrandurchtritt von unmarkierten bioorganischen Molekülen direkt und in Echtzeit mittels Fluoreszenz zu verfolgen. Dieser In-vitro-Assay kann auf unterschiedliche Kanalproteine und Analyten angewendet werden, eignet sich zur schnellen Charakterisierung von Kanalmodulatoren und liefert zudem die absolute Kinetik der Translokation. Mit dieser neuen biophysikalischen Methode konnten wir erstmalig die direkte schnelle Translokation des antibiotisch wirksamen Peptids Protamin durch das bakterielle Transmembranprotein OmpF nachweisen.
Angewandte Chemie International Edition, 2014
The efficacy of drugs and biomolecules relies on their ability to pass through the bilayer. The d... more The efficacy of drugs and biomolecules relies on their ability to pass through the bilayer. The development of methods to directly and sensitively monitor these membrane transport processes has remained an experimental challenge. A macrocyclic host (p-sulfonatocalix[4]arene or cucurbit[7]uril) and a fluorescent dye (lucigenin or berberine) are encapsulated as a chemosensing ensemble inside liposomes, which allows for a direct, real-time fluorescence monitoring of the passage of unlabeled bioorganic analytes. This in vitro assay is transferable to different channel proteins and analytes, has potential for fluorescence-based screening, e.g., of channel modulators, and yields the absolute kinetics of translocation. Using this new biophysical method, we observed for the first time direct rapid translocation of protamine, an antimicrobial peptide, through the bacterial transmembrane protein OmpF.
The journal of physical chemistry. B, Jan 8, 2015
Coulomb repulsion between like-charged side chains is presently viewed as a major force that impa... more Coulomb repulsion between like-charged side chains is presently viewed as a major force that impacts the biological activity of intrinsically disordered polypeptides (IDPs) by determining their spatial dimensions. We investigated short synthetic models of IDPs, purely composed of ionizable amino acid residues and therefore expected to display an extreme structural and dynamic response to pH variation. Two synergistic, custom-made, time-resolved fluorescence methods were applied in tandem to study the structure and dynamics of the acidic and basic hexapeptides Asp6, Glu6, Arg6, Lys6, and His6 between pH 1 and 12. (i) End-to-end distances were obtained from the short-distance Förster resonance energy transfer (sdFRET) from N-terminal 5-fluoro-l-tryptophan (FTrp) to C-terminal Dbo. (ii) End-to-end collision rates were obtained for the same peptides from the collision-induced fluorescence quenching (CIFQ) of Dbo by FTrp. Unexpectedly, the very high increase of charge density at elevated...
Journal of Biomolecular Nmr, 2003
For a detailed NMR study of the dynamics of the cold shock protein CspB from Bacillus subtilis, w... more For a detailed NMR study of the dynamics of the cold shock protein CspB from Bacillus subtilis, we determined (15)N transverse and longitudinal relaxation rates and heteronuclear nuclear Overhauser effects at different solvent viscosities. Up to a relative viscosity of 2, which is equivalent to 27% ethylene glycol (EG), the overall correlation time follows the linear Stokes-Einstein equation. At a relative viscosity of 6 (70% EG) the correlation time deviates from linearity by 30%, indicating that CspB tumbles at a higher rate as expected from the solvent viscosity probably due to a preferential binding of water molecules at the protein surface. The corresponding hydrodynamic radii, determined by NMR diffusion experiments, show no variation with viscosity. The amplitudes of intramolecular motions on a sub-nanosecond time scale revealed by an extended Lipari-Szabo analysis were mainly independent of the solvent viscosity. The lower limit of the NMR 'observation window' for the internal correlation time shifts above 0.5 ns at 70% EG, which is directly reflected in the experimentally derived internal correlation times. Chemical exchange contributions to the transverse relaxation rates derived from the Lipari-Szabo approach coincide with the experimentally determined values from the transverse (1)H-(15)N dipolar/(15)N chemical shift anisotropy relaxation interference. These contributions originate from fast protein folding reactions on a millisecond timescale, which get retarded at increased solvent viscosities.
Proceedings of the National Academy of Sciences of the United States of America, May 1, 1997
The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds ... more The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds extremely fast in a reversible N leftrightharpoons U two-state reaction. Both unfolding and refolding are strongly decelerated when the viscosity of the solvent is increased by adding ethylene glycol or sucrose. The folding of CspB thus seems to follow Kramers' model for reactions in which the reactants must diffuse together. It indicates that the compaction of the protein chain occurs in the rate-limiting step of folding. Chain diffusion to a productively collapsed form and the crossing of a high energy barrier are thus tightly coupled in this folding reaction, and the measured reaction rate depends on both the diffusion of the protein chain in the solvent and the magnitude of the activation energy. We suggest that in protein folding an energetic barrier is essential to separate the native from the unfolded conformations of a protein. This barrier protects the ordered structure of a native protein against continuous unfolding by diffusive chain motions and leads to apparent two-state behavior.
Journal of biomolecular NMR, 2003
For a detailed NMR study of the dynamics of the cold shock protein CspB from Bacillus subtilis, w... more For a detailed NMR study of the dynamics of the cold shock protein CspB from Bacillus subtilis, we determined (15)N transverse and longitudinal relaxation rates and heteronuclear nuclear Overhauser effects at different solvent viscosities. Up to a relative viscosity of 2, which is equivalent to 27% ethylene glycol (EG), the overall correlation time follows the linear Stokes-Einstein equation. At a relative viscosity of 6 (70% EG) the correlation time deviates from linearity by 30%, indicating that CspB tumbles at a higher rate as expected from the solvent viscosity probably due to a preferential binding of water molecules at the protein surface. The corresponding hydrodynamic radii, determined by NMR diffusion experiments, show no variation with viscosity. The amplitudes of intramolecular motions on a sub-nanosecond time scale revealed by an extended Lipari-Szabo analysis were mainly independent of the solvent viscosity. The lower limit of the NMR 'observation window' for th...
Nature structural biology, 1999
There has been some debate as to whether protein folding involves diffusive chain motions and thu... more There has been some debate as to whether protein folding involves diffusive chain motions and thus depends on solvent viscosity. The interpretation of folding kinetics in viscous solvents has remained difficult and controversial, in that viscogenic agents affect folding rates not only by increasing solvent viscosity but also by increasing protein stability. By carefully choosing experimental conditions, we can now eliminate the effect on stability and show that the folding dynamics of the cold shock protein CspB are viscosity dependent. Thus Kramers' theory of reaction rates rather than transition state theory should be used to describe this folding reaction.
Schweitzer-Stenner/Peptide Folding, Misfolding, and Nonfold, 2012
The Journal of Physical Chemistry B, 2013
The structural and dynamic properties of a flexible peptidic chain codetermine its biological act... more The structural and dynamic properties of a flexible peptidic chain codetermine its biological activity. These properties are imprinted in intrachain site-to-site distances as well as in diffusion coefficients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of Forster resonance energy transfer (FRET) between two chain sites labeled with a FRET donor and acceptor. Both could be obtained from time-resolved FRET measurements if their individual contributions to the FRET efficiency could be systematically varied. Because the FRET diffusion enhancement (FDE) depends on the donor-fluorescence lifetime, it has been proposed that the FDE can be reduced by shortening the donor lifetime through an external quencher. Benefiting from the high diffusion sensitivity of short-distance FRET, we tested this concept experimentally on a (Gly−Ser) 6 segment labeled with the donor/acceptor pair naphthylalanine/2,3-diazabicyclo[2.2.2]oct-2-ene (NAla/Dbo). Surprisingly, the very effective quencher potassium iodide (KI) had no effect at all on the average donor−acceptor distance, although the donor lifetime was shortened from ca. 36 ns in the absence of KI to ca. 3 ns in the presence of 30 mM KI. We show that the proposed approach had to fail because it is not the experimentally observed but the radiative donor lifetime that controls the FDE. Because of that, any FRET ensemble measurement can easily underestimate diffusion and might be misleading even if it employs the Haas−Steinberg diffusion equation (HSE). An extension of traditional FRET analysis allowed us to evaluate HSE simulations and to corroborate as well as generalize the experimental results. We demonstrate that diffusion-enhanced FRET depends on the radiative donor lifetime as it depends on the diffusion coefficient, a useful symmetry that can directly be applied to distinguish dynamic and structural effects of viscous cosolvents on the polymer chain. We demonstrate that the effective FRET rate and the recovered donor−acceptor distance depend on the quantum yield, most strongly in the absence of diffusion, which has to be accounted for in the interpretation of distance trends monitored by FRET. . Selected donor−acceptor and donor-only labeled peptides composed of Gly−Ser units. Donors are naphthyl-1-L-alanine (NAla) and 5fluoro-L-tryptophan (FTrp); the acceptor is Dbo, whose optically active group is the azo group of the bicyclic chromophore.
Proceedings of the National Academy of Sciences, 1997
The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds ... more The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds extremely fast in a reversible N ª U two-state reaction. Both unfolding and refolding are strongly decelerated when the viscosity of the solvent is increased by adding ethylene glycol or sucrose.
Journal of Molecular Biology, 2009
Most globular protein chains, when transferred from high to low denaturant concentrations, collap... more Most globular protein chains, when transferred from high to low denaturant concentrations, collapse instantly before they refold to their native state. The initial compaction of the protein molecule is assumed to have a key effect on the folding pathway, but it is not known whether the earliest structures formed during or instantly after collapse are defined by local or by non-local interactions-that is, by secondary structural elements or by loop closure of long segments of the protein chain. Stable closure of one or several long loops can reduce the chain entropy at a very early stage and can prevent the protein from following non-productive pathways whose number grows exponentially with the length of the protein chain. In Escherichia coli adenylate kinase (AK), about seven long loops define the topology of the native structure. We selected four loop-forming sections of the chain and probed the time course of loop formation during refolding of AK. We labeled the termini of the loop segments with tryptophan and cysteine-5-amidosalicylic acid. This donor-acceptor pair of probes used with fluorescence resonance excitation energy transfer spectroscopy (FRET) is suitable for detecting very short distances and thus is able to distinguish between random and specific compactions. Refolding of AK was initiated by stopped-flow mixing, followed simultaneously by donor and acceptor fluorescence, and analyzed in terms of energy transfer efficiency and distance. In the collapsed state of AK, observed after the 5-ms dead time of the instrument, one of the selected segments shows a native-like separation of its termini; it forms a loop already in the collapsed state. A second segment that includes the first but is longer by 15 residues shows an almost native-like separation of its termini. In contrast, a segment that is shorter but part of the second segment shows a distance separation of its termini as high as a segment that spans almost the whole protein chain. We conclude that a specific network of non-local interactions, the closure of one or several loops, can play an important role in determining the protein folding pathway at its early phases. . † T. Orevi and E. B. Ishai contributed equally to this work. Abbreviations used: A-AK, AK labeled with a FRET acceptor probe; D-AK, AK labeled with an engineered Trp residue as a FRET donor probe; DA-AK, AK labeled with both donor and acceptor; ETE, energy transfer efficiency; FRET, fluorescence resonance excitation energy transfer; GndHCl, guanidinium hydrochloride; SA, 5-amidosalicylic acid; tr-FRET, time-resolved FRET; wt-AK, Escherichia coli C77S (wild type) adenylate kinase.
Journal of Molecular Biology, 2002
The cold-shock protein CspB folds rapidly in a N Y U two-state reaction via a transition state th... more The cold-shock protein CspB folds rapidly in a N Y U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive ðDV ‡ NU ¼ 16ð^4Þ ml=molÞ and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well ðDV ‡ UN ¼ 53ð^6Þ ml=molÞ and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, DV ‡ UN decreases significantly, suggesting that compact unfolded forms become populated under these conditions. Abbreviations used: CspB, cold-shock protein from Bacillus subtilis; GdmSCN, guanidinium thiocyanate; l, measured rate of a reaction; k ij , microscopic rate constant; DV ‡ app , apparent activation volume; DV ‡ ij , microscopic activation volume; n, n ij , denaturant-dependence of reaction and activation volumes.
Biophysical Chemistry, 2002
The folding reactions of several proteins are well described as diffusional barrier crossing proc... more The folding reactions of several proteins are well described as diffusional barrier crossing processes, which suggests that they should be analyzed by Kramers' rate theory rather than by transition state theory. For the cold shock protein Bc-Csp from Bacillus caldolyticus, we measured stability and folding kinetics, as well as solvent viscosity as a function of temperature and denaturant concentration. Our analysis indicates that diffusional folding reactions can be treated by transition state theory, provided that the temperature and denaturant dependence of the solvent viscosity is properly accounted for, either at the level of the measured rate constants or of the calculated activation parameters. After viscosity correction the activation barriers for folding become less enthalpic and more entropic. The transition from an enthalpic to an entropic folding barrier with increasing temperature is, however, apparent in the data before and after this correction. It is a consequence of the negative activation heat capacity of refolding, which is independent of solvent viscosity. Bc-Csp and its mesophilic homolog Bs-CspB from Bacillus subtilis differ strongly in stability but show identical enthalpic and entropic barriers to refolding. The increased stability of Bc-Csp originates from additional enthalpic interactions that are established after passage through the activated state. As a consequence, the activation enthalpy of unfolding is increased relative to Bs-CspB. ᮊ
Biochemistry, 1999
A protein chain must move relative to the solvent molecules and explore many conformations when i... more A protein chain must move relative to the solvent molecules and explore many conformations when it folds from the extended unfolded state to the compact native state. Experimental and theoretical approaches suggest that diffusional processes in fact contribute to the kinetics of protein folding. We describe here how variations of the solvent viscosity can be employed to uncover the diffusional contributions to a folding reaction and assess the use of transition state theory and Kramers' rate theory for the analysis of protein folding reactions.
Biochemistry, 1999
A pressure-jump apparatus was employed in investigating the kinetics of protein unfolding and ref... more A pressure-jump apparatus was employed in investigating the kinetics of protein unfolding and refolding. In the reaction cell, the pressure can be increased or decreased by 100-160 bar within 50-100 microseconds and then held constant. Thus, unfolding and refolding reactions in the time range from 70 microseconds to 70 s can be followed with this technique. Measurements are possible in the transition regions of thermally or denaturant-induced folding in a wide range of temperatures and solvent conditions. We used this pressure-jump method to determine the temperature dependence of the rate constants of unfolding and refolding of the cold shock protein of Bacillus subtilis and of three variants thereof with Phe --> Ala substitutions in the central beta-sheet region. For all variants, the change in heat capacity occurred in refolding between the unfolded and activated states, suggesting that the overall native-like character of the activated state of folding was not changed by the deletion of individual Phe side chains. The Phe27Ala mutation affected the rate of unfolding only; the Phe15Ala and Phe17Ala mutations changed the kinetics of both unfolding and refolding. Although the activated state of folding of the cold shock protein is overall native-like, individual side chains are still in a non-native environment.
Biochemistry, 2005
A variety of biophysical methods used to study proteins requires protein modification using conju... more A variety of biophysical methods used to study proteins requires protein modification using conjugated molecular probes. Cysteine is the main residue that can be modified without the risk of altering other residues in the protein chain. It is possible to label several cysteines in a protein using highly selective labeling reactions, if the cysteines react at very different rates. The reactivity of a cysteine residue introduced into an exposed surface site depends on the fraction of cysteine in the deprotonated state. Here, it is shown that cysteine reactivity differences can be effectively predicted by an electrostatic model that yields site-specifically the fractions of cysteinate. The model accounts for electrostatic interactions between the cysteinyl anion and side chains, the local protein backbone, and water. The energies of interaction with side chains and the main chain are calculated by using the two different dielectric constants, 40 and 22, respectively. Twenty-six mutants of Escherichia coli adenylate kinase were produced, each containing a single cysteine at the protein surface, and the rates of the reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) were measured. Cysteine residues were chosen on the basis of locations that were expected to allow modification of the protein with minimal risk of perturbing its structure. The reaction rates spanned a range of 6 orders of magnitude. The correlation between predicted fractions of cysteinate and measured reaction rates was strong (R = 92%) and especially high (R = 97%) for cysteines at the helix termini. The approach developed here allows reasonably fast, automated screening of protein surfaces to identify sites that permit efficient preparations of double- or triple-labeled protein.
Angewandte Chemie, 2014
ABSTRACT Die Effektivität von Wirkstoffen und Biomolekülen basiert auf ihrer Fähigkeit, die Lipid... more ABSTRACT Die Effektivität von Wirkstoffen und Biomolekülen basiert auf ihrer Fähigkeit, die Lipidmembran zu passieren. Die Entwicklung von Methoden, diese Transportprozesse direkt und mit hoher Empfindlichkeit zu verfolgen, bleibt eine Herausforderung. Der Einschluss eines chemosensorischen Ensembles, bestehend aus einem makrocyclischen Wirt (p-Sulfonatocalix[4]aren oder Cucurbit[7]uril) und einem Fluoreszenzfarbstoff (Lucigenin oder Berberin) in Liposomen ermöglicht es, den Membrandurchtritt von unmarkierten bioorganischen Molekülen direkt und in Echtzeit mittels Fluoreszenz zu verfolgen. Dieser In-vitro-Assay kann auf unterschiedliche Kanalproteine und Analyten angewendet werden, eignet sich zur schnellen Charakterisierung von Kanalmodulatoren und liefert zudem die absolute Kinetik der Translokation. Mit dieser neuen biophysikalischen Methode konnten wir erstmalig die direkte schnelle Translokation des antibiotisch wirksamen Peptids Protamin durch das bakterielle Transmembranprotein OmpF nachweisen.
Angewandte Chemie International Edition, 2014
The efficacy of drugs and biomolecules relies on their ability to pass through the bilayer. The d... more The efficacy of drugs and biomolecules relies on their ability to pass through the bilayer. The development of methods to directly and sensitively monitor these membrane transport processes has remained an experimental challenge. A macrocyclic host (p-sulfonatocalix[4]arene or cucurbit[7]uril) and a fluorescent dye (lucigenin or berberine) are encapsulated as a chemosensing ensemble inside liposomes, which allows for a direct, real-time fluorescence monitoring of the passage of unlabeled bioorganic analytes. This in vitro assay is transferable to different channel proteins and analytes, has potential for fluorescence-based screening, e.g., of channel modulators, and yields the absolute kinetics of translocation. Using this new biophysical method, we observed for the first time direct rapid translocation of protamine, an antimicrobial peptide, through the bacterial transmembrane protein OmpF.
The journal of physical chemistry. B, Jan 8, 2015
Coulomb repulsion between like-charged side chains is presently viewed as a major force that impa... more Coulomb repulsion between like-charged side chains is presently viewed as a major force that impacts the biological activity of intrinsically disordered polypeptides (IDPs) by determining their spatial dimensions. We investigated short synthetic models of IDPs, purely composed of ionizable amino acid residues and therefore expected to display an extreme structural and dynamic response to pH variation. Two synergistic, custom-made, time-resolved fluorescence methods were applied in tandem to study the structure and dynamics of the acidic and basic hexapeptides Asp6, Glu6, Arg6, Lys6, and His6 between pH 1 and 12. (i) End-to-end distances were obtained from the short-distance Förster resonance energy transfer (sdFRET) from N-terminal 5-fluoro-l-tryptophan (FTrp) to C-terminal Dbo. (ii) End-to-end collision rates were obtained for the same peptides from the collision-induced fluorescence quenching (CIFQ) of Dbo by FTrp. Unexpectedly, the very high increase of charge density at elevated...