-
[show abstract]
[hide abstract]
ABSTRACT: Protein folding coupled to binding of a specific ligand is frequently observed in biological processes. In recent years numerous studies have addressed the structural properties of the unfolded proteins in the absence of their ligands. Surprisingly few time-resolved investigations on coupled folding and binding reactions have been published up to date and the dynamics and kinetic mechanisms of these processes are still only poorly understood. Especially, it is still unsolved for most systems which conformation of the protein is recognized by the ligand (conformational selection vs. folding-after-binding) and whether the ligand influences the folding kinetics. Here we review experimental methods, kinetic models and time-resolved experimental studies of coupled folding and binding reactions.
Current Opinion in Structural Biology 11/2011; 22(1):21-9. · 9.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Understanding the mechanism of protein folding requires a detailed knowledge of the structural properties of the barriers separating unfolded from native conformations. The S-peptide from ribonuclease S forms its α-helical structure only upon binding to the folded S-protein. We characterized the transition state for this binding-induced folding reaction at high resolution by determining the effect of site-specific backbone thioxylation and side-chain modifications on the kinetics and thermodynamics of the reaction, which allows us to monitor formation of backbone hydrogen bonds and side-chain interactions in the transition state. The experiments reveal that α-helical structure in the S-peptide is absent in the transition state of binding. Recognition between the unfolded S-peptide and the S-protein is mediated by loosely packed hydrophobic side-chain interactions in two well defined regions on the S-peptide. Close packing and helix formation occurs rapidly after binding. Introducing hydrophobic residues at positions outside the recognition region can drastically slow down association.
Proceedings of the National Academy of Sciences 02/2011; 108(10):3952-7. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A reversible structural unlocking reaction, in which the close-packed van der Waals interactions break cooperatively, has been found for the villin headpiece subdomain (HP35) using triplet-triplet-energy transfer to monitor conformational fluctuations from equilibrium. Unlocking is associated with an unfavorable enthalpy change (DeltaH(0) = 35 +/- 4 kJ/mol) which is nearly compensated in free energy by the entropy change (DeltaS(0) = 112 +/- 20 Jxmol(-1)xK(-1)). The unlocking reaction has a time constant of about 1 mus at 5 degrees C and is enthalpy-limited with an activation energy of 32 +/- 1 kJ/mol and a large Arrhenius preexponential factor of A = 7.5 x 10(11) s(-1). In the unlocked state a fast local conformational fluctuation with a time constant of 170 ns and a low activation barrier of 17 +/- 1 kJ/mol leads to unfolding of the C-terminal helix and to its undocking from the core. On a much slower time scale, global unfolding occurs from the unlocked state. These results suggest that native protein structures are locked into conformations with low amplitude motions. Large scale motions and global unfolding require an initial structural unlocking step leading to a state with properties of a dry molten globule. The experiments additionally yielded information on the dynamics of loop formation between different positions in unfolded HP35. Comparison of the results with dynamics in unstructured model peptides indicates slightly decelerated kinetics of local loop formation in the C-terminal region which points at residual, nonrandom structure. Dynamics of long-range loop formation, in contrast, are not influenced by residual structure, which argues against unfolded state properties as molecular origin for ultrafast folding of HP35.
Proceedings of the National Academy of Sciences 03/2010; 107(11):4955-60. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Efficient formation of specific intermolecular interactions is essential for self-assembly of biological structures. The foldon domain is an evolutionarily optimized trimerization module required for assembly of the large, trimeric structural protein fibritin from phage T4. Monomers consisting of the 27 amino acids comprising a single foldon domain subunit spontaneously form a natively folded trimer. During assembly of the foldon domain, a monomeric intermediate is formed on the submillisecond time scale, which provides the basis for two consecutive very fast association reactions. Mutation of an intermolecular salt bridge leads to a monomeric protein that resembles the kinetic intermediate in its spectroscopic properties. NMR spectroscopy revealed essentially native topology of the monomeric intermediate with defined hydrogen bonds and side-chain interactions but largely reduced stability compared to the native trimer. This structural preorganization leads to an asymmetric charge distribution on the surface that can direct rapid subunit recognition. The low stability of the intermediate allows a large free-energy gain upon trimerization, which serves as driving force for rapid assembly. These results indicate different free-energy landscapes for folding of small oligomeric proteins compared to monomeric proteins, which typically avoid the transient population of intermediates.
Journal of Molecular Biology 05/2009; 389(1):103-14. · 4.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Coupling fast triplet-triplet energy transfer (TTET) between xanthone and naphthylalanine to the helix-coil equilibrium in alanine-based peptides allowed the observation of local equilibrium fluctuations in alpha-helices on the nanoseconds to microseconds time scale. The experiments revealed faster helix unfolding in the terminal regions compared with the central parts of the helix with time constants varying from 250 ns to 1.4 micros at 5 degrees C. Local helix formation occurs with a time constant of approximately 400 ns, independent of the position in the helix. Comparing the experimental data with simulations using a kinetic Ising model showed that the experimentally observed dynamics can be explained by a 1-dimensional boundary diffusion with position-independent elementary time constants of approximately 50 ns for the addition and of approximately 65 ns for the removal of an alpha-helical segment. The elementary time constant for helix growth agrees well with previously measured time constants for formation of short loops in unfolded polypeptide chains, suggesting that helix elongation is mainly limited by a conformational search.
Proceedings of the National Academy of Sciences 02/2009; 106(4):1057-62. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Thioxoamide (thioamide) bonds are nearly isosteric substitutions for amides but have altered hydrogen-bonding and photophysical properties. They are thus well-suited backbone modifications for physicochemical studies on peptides and proteins. The effect of thioxoamides on protein structure and stability has not been subject to detailed experimental investigations up to date. We used alanine-based model peptides to test the influence of single thioxoamide bonds on alpha-helix structure and stability. The results from circular dichroism measurements show that thioxoamides are strongly helix-destabilizing. The effect of an oxo-to-thioxoamide backbone substitution is of similar magnitude as an alanine-to-glycine substitution resulting in a helix destabilization of about 7 kJ/mol. NMR characterization of a helical peptide with a thioxopeptide bond near the N-terminus indicates that the thioxopeptide moiety is tolerated in helical structures. The thioxoamide group is engaged in an i, i+4 hydrogen bond, arguing against the formation of a 3(10)-helical structure as suggested for the N-termini of alpha-helices in general and for thioxopeptides in particular.
Journal of the American Chemical Society 07/2008; 130(25):8079-84. · 9.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In this report, it is shown by a combination of stopped-flow CD, fluorescence, and time-resolved NMR studies that the Ca(2+)-induced refolding of bovine alpha-lactalbumin (BLA) at constant denaturant concentration (4 M urea) exhibits triple-exponential kinetics. In order to distinguish between parallel folding pathways and a strictly sequential formation of the native state, interrupted refolding experiments were conducted. We show here that the Ca(2+)-induced refolding of BLA involves parallel pathways and the transient formation of a folding intermediate on the millisecond timescale. Our data furthermore suggest that the two structurally homologous proteins BLA and hen egg white lysozyme share a common folding mechanism. We provide evidence that the guiding role of long-range interactions in the unfolded state of lysozyme in mediating intersubdomain interactions during folding is replaced in the case of BLA by the Ca(2+)-binding site. Time-resolved NMR spectroscopy, in combination with fast ion release from caged compounds, enables the measurement of complex protein folding kinetics at protein concentrations as low as 100 microM and the concomitant detection of conformational transitions with rate constants of up to 8 s(-1).
Journal of Molecular Biology 06/2008; 378(3):686-98. · 4.00 Impact Factor
-
01/2008: pages 377 - 410; , ISBN: 9783527619498
-
01/2008: pages 809 - 855; , ISBN: 9783527619498
-
01/2008: pages 411 - 444; , ISBN: 9783527619498
-
[show abstract]
[hide abstract]
ABSTRACT: psi[CS-NH]4-RNase S, a site specific modified version of RNase S obtained by thioxylation (O/S exchange) at the Ala4-Ala5- peptide bond, was used to evaluate the impact of protein backbone photoswitching on bioactivity. psi[CS-NH](4)-RNase S was yielded by recombination of the S-protein and the respective chemically synthesized thioxylated S-peptide derivative. Comparison with RNase S revealed similar thermodynamic stability of the complex and an unperturbed enzymatic activity toward cytidine 2',3'-cyclic monophosphate (cCMP). Reversible photoisomerization with a highly increased cis/trans isomer ratio of the thioxopeptide bond of psi[CS-NH](4)-RNase S in the photostationary state occurred under UV irradiation conditions (254 nm). The slow thermal reisomerization (t(1/2) = 180 s) permitted us to determine the enzymatic activity of cis psi[CS-NH](4)-RNase S by measurement of initial rates of cCMP hydrolysis. Despite thermodynamic stability of cis psi[CS-NH](4)-RNase S, its enzymatic activity is completely abolished but recovers after reisomerization. We conclude that the thioxopeptide bond modified polypeptide backbone represents a versatile probe for site-directed photoswitching of proteins.
Journal of the American Chemical Society 04/2007; 129(16):4910-8. · 9.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The conformational search for favorable intramolecular interactions during protein folding is limited by intrachain diffusion processes. Recent studies on the dynamics of loop formation in unfolded polypeptide chains have focused on loops involving residues near the chain ends. During protein folding, however, most contacts are formed between residues in the interior of the chain. We compared the kinetics of end-to-end loop formation (type I loops) to the formation of end-to-interior (type II loops) and interior-to-interior loops (type III loops) using triplet-triplet energy transfer from xanthone to naphthylalanine. The results show that formation of type II and type III loops is slower compared to type I loops of the same size and amino acid sequence. The rate constant for type II loop formation decreases with increasing overall chain dimensions up to a limiting value, at which loop formation is about 2.5-fold slower for type II loops compared to type I loops. Comparing type II loops of different loop size and amino acid sequence shows that the ratio of loop dimension over total chain dimension determines the rate constant for loop formation. Formation of type III loops is 1.7-fold slower than formation of type II loops, indicating that local chain motions are strongly coupled to motions of other chain segments which leads to faster dynamics toward the chain ends. Our results show that differences in the kinetics of formation of type I, type II, and type III loops are mainly caused by differences in internal flexibility at the different positions in the polypeptide chain. Interactions of the polypeptide chain with the solvent contribute to the kinetics of loop formation, which are strongly viscosity-dependent. However, the observed differences in the kinetics of formation of type I, type II, and type III loops are not due to the increased number of peptide-solvent interactions in type II and type III loops compared to type I loops as indicated by identical viscosity dependencies for the kinetics of formation of the different types of loops.
Journal of the American Chemical Society 02/2007; 129(3):672-9. · 9.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Intrachain diffusion processes play an important role in protein folding and function. In this chapter we discuss the application of triplet-triplet energy transfer to directly measure rate constants for intrachain contact formation in polypeptide chains. The photochemistry of triplet-triplet energy transfer is described, experimental prerequisites of the method are discussed, and a detailed description of the experimental protocols and data analysis is given.
Methods in molecular biology (Clifton, N.J.) 02/2007; 350:169-87.
-
[show abstract]
[hide abstract]
ABSTRACT: Intrachain loop formation allows unfolded polypeptide chains to search for favorable interactions during protein folding. We applied triplet-triplet energy transfer between a xanthone moiety and naphthylalanine to directly measure loop formation in various unfolded polypeptide chains with loop regions consisting of polyserine, poly(glycine-serine) or polyproline. By combination of femtosecond and nanosecond laserflash experiments loop formation could be studied over many orders of magnitude in time from picoseconds to microseconds. The results reveal processes on different time scales indicating motions on different hierarchical levels of the free energy surface. A minor (<15%) very fast reaction with a time constant of approximately 3 ps indicates equilibrium conformations with donor and acceptor in contact at the time of the laserflash. Complex kinetics of loop formation were observed on the 50- to 500-ps time scale, which indicate motions within a local well on the energy landscape. Conformations within this well can form loops by undergoing local motions without having to cross major barriers. Exponential kinetics observed on the 10- to 100-ns time scale are caused by diffusional processes involving large-scale motions that allow the polypeptide chain to explore the complete conformational space. These results indicate that the free energy landscape for unfolded polypeptide chains and native proteins have similar properties. The presence of local energy minima reduces the conformational space and accelerates the conformational search for energetically favorable local intrachain contacts.
Proceedings of the National Academy of Sciences 02/2007; 104(7):2163-8. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Characterization of the unfolded state is essential for the understanding of the protein folding reaction. We performed time-resolved FRET measurements to gain information on the dimensions and the internal dynamics of unfolded polypeptide chains. Using an approach based on global analysis of data obtained from two different donor-acceptor pairs allowed for the determination of distance distribution functions and diffusion constants between the chromophores. Results on a polypeptide chain consisting of 16 Gly-Ser repeats between the FRET chromophores reveal an increase in the average end-to-end distance from 18.9 to 39.2 Angstrom between 0 and 8 M GdmCl. The increase in chain dimensions is accompanied by an increase in the end-to-end diffusion constant from (3.6 +/- 1.0) x 10(-7) cm(2) s(-1) in water to (14.8 +/- 2.5) x 10(-7) cm(2) s(-1) in 8 M GdmCl. This finding suggests that intrachain interactions in water exist even in very flexible chains lacking hydrophobic groups, which indicates intramolecular hydrogen bond formation. The interactions are broken upon denaturant binding, which leads to increased chain flexibility and longer average end-to-end distances. This finding implies that rapid collapse of polypeptide chains during refolding of denaturant-unfolded proteins is an intrinsic property of polypeptide chains and can, at least in part, be ascribed to nonspecific intramolecular hydrogen bonding. Despite decreased intrachain diffusion constants, the conformational search is accelerated in the collapsed state because of shorter diffusion distances. The measured distance distribution functions and diffusion constants in combination with Szabo-Schulten-Schulten theory were able to reproduce experimentally determined rate constants for end-to-end loop formation.
Proceedings of the National Academy of Sciences 09/2006; 103(33):12394-9. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The characterization of the free energy barriers has been a major goal in studies on the mechanism of protein folding. Testing the effect of mutations or denaturants on protein folding reactions revealed that transition state movement is rare, suggesting that folding barriers are robust and narrow maxima on the free energy landscape. Here we demonstrate that the application of multiple perturbations allows the observation of small transition state movements that escape detection in single perturbation experiments. We used tendamistat as a model protein to test the broadness of the free energy barriers. Tendamistat folds over two consecutive transition states and through a high-energy intermediate. Measuring the combined effect of temperature and denaturant on the position of the transition state in the wild-type protein and in several mutants revealed that the early transition state shows significant transition state movement. Its accessible surface area state becomes more native-like with destabilization of the native state by temperature. To the same extent, the entropy of the early transition state becomes more native-like with increasing denaturant concentration, in accordance with Hammond behavior. The position of the late transition state, in contrast, is much less sensitive to the applied perturbations. These results suggest that the barriers in protein folding become increasingly narrow as the folding polypeptide chain approaches the native state.
Journal of Molecular Biology 04/2006; 357(2):655-64. · 4.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Collagen consists of repetitive Gly-Xaa-Yaa tripeptide units with proline and hydroxyproline frequently found in the Xaa and Yaa position, respectively. This sequence motif allows the formation of a highly regular triple helix that is stabilized by steric (entropic) restrictions in the constituent polyproline-II-helices and backbone hydrogen bonds between the three strands. Concentration-dependent association reactions and slow prolyl isomerization steps have been identified as major rate-limiting processes during collagen folding. To gain information on the dynamics of triple-helix formation in the absence of these slow reactions, we performed stopped-flow double-jump experiments on cross-linked fragments derived from human type III collagen. This technique allowed us to measure concentration-independent folding kinetics starting from unfolded chains with all peptide bonds in the trans conformation. The results show that triple-helix formation occurs with a rate constant of 113 +/- 20 s(-1) at 3.7 degrees C and is virtually independent of temperature, indicating a purely entropic barrier. Comparison of the effect of guanidinium chloride on folding kinetics and stability reveals that the rate-limiting step is represented by bringing 10 consecutive tripeptide units (3.3 per strand) into a triple-helical conformation. The following addition of tripeptide units occurs on a much faster time scale and cannot be observed experimentally. These results support an entropy-controlled zipper-like nucleation/growth mechanism for collagen triple-helix formation.
Proceedings of the National Academy of Sciences 10/2005; 102(39):13897-902. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The structural characterization of transition states is essential for understanding the mechanism of protein folding. Analyzing the effect of mutations on protein stability and folding kinetics in phi-value analysis is commonly used to gain information about the presence of side-chain interactions in transition states. Recently, specific binding of ligands to engineered binding sites was applied to monitor the formation of local structures in transition states (psi analysis). A surprising result from psi analysis was the presence of parallel folding pathways in all reported studies and a major discrepancy between phi and psi values measured in the same protein. Here, we show that psi values cannot be analyzed in the same way as other rate-equilibrium free energy relationships due to the involvement of bimolecular reactions that may have different dissociation constants for the native, unfolded and transition state. As a consequence, psi values reflect the relative binding energy (kappa) of the transition state only for the extreme values of kappa=0 or kappa=1. In all other cases, non-linear rate-equilibrium free-energy relationships (Leffler plots) are observed. This apparently indicates the presence of parallel folding pathways even if folding occurs over a single homogeneous transition state. Consequently, the results from Leffler plots do not yield information about the structural properties of the transition state. This explains the lack of agreement between results from psi analysis and other methods used to characterize protein folding transition states. We further show that the same considerations apply for the analysis of the effect of pH on protein folding.
Journal of Molecular Biology 09/2005; 351(2):393-401. · 4.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Glycine and proline residues are frequently found in turn and loop structures of proteins and are believed to play an important role during chain compaction early in folding. We investigated their effect on the dynamics of intrachain loop formation in various unstructured polypeptide chains. Loop formation is significantly slower around trans prolyl peptide bonds and faster around glycine residues compared to any other amino acid. However, short loops are formed fastest around cis prolyl bonds with a time constant of 6 ns for end-to-end contact formation in a four-residue loop. Formation of short loops encounters activation energies in the range of 15 to 30 kJ/mol. The altered dynamics around glycine and trans prolyl bonds can be mainly ascribed to their effects on the activation energy. The fast dynamics around cis prolyl bonds, in contrast, originate in a higher Arrhenius pre-exponential factor, which compensates for an increased activation energy for loop formation compared to trans isomers. All-atom simulations of proline-containing peptides indicate that the conformational space for cis prolyl isomers is largely restricted compared to trans isomers. This leads to decreased average end-to-end distances and to a smaller loss in conformational entropy upon loop formation in cis isomers. The results further show that glycine and proline residues only influence formation of short loops containing between 2 and 10 residues, which is the typical loop size in native proteins. Formation of larger loops is not affected by the presence of a single glycine or proline residue.
Journal of the American Chemical Society 04/2005; 127(10):3346-52. · 9.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Chemical denaturants are frequently used to unfold proteins and to characterize mechanisms and transition states of protein folding reactions. The molecular basis of the effect of urea and guanidinium chloride (GdmCl) on polypeptide chains is still not well understood. Models for denaturant--protein interaction include both direct binding and indirect changes in solvent properties. Here we report studies on the effect of urea and GdmCl on the rate constants (k(c)) of end-to-end diffusion in unstructured poly(glycine-serine) chains of different length. Urea and GdmCl both lead to a linear decrease of lnk(c) with denaturant concentration, as observed for the rate constants for protein folding. This suggests that the effect of denaturants on chain dynamics significantly contributes to the denaturant-dependence of folding rate constants for small proteins. We show that this linear dependency is the result of two additive non-linear effects, namely increased solvent viscosity and denaturant binding. The contribution from denaturant binding can be quantitatively described by Schellman's weak binding model with binding constants (K) of 0.62(+/-0.01)M(-1) for GdmCl and 0.26(+/-0.01)M(-1) for urea. In our model peptides the number of binding sites and the effect of a bound denaturant molecule on chain dynamics is identical for urea and GdmCl. The results further identify the polypeptide backbone as the major denaturant binding site and give an upper limit of a few nanoseconds for residence times of denaturant molecules on the polypeptide chain.
Journal of Molecular Biology 02/2005; 345(1):153-62. · 4.00 Impact Factor
