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Dynamics and function of proteins are governed by the structural and energetic properties of the different states they adopt and the barriers separating them. In earlier work, native-state triplet–triplet energy transfer (TTET) on the villin headpiece subdomain (HP35) revealed an equilibrium between a locked native state and an unlocked native state, which are structurally similar but have different dynamic properties. The locked state is restricted to low amplitude motions, whereas the unlocked state shows increased conformational flexibility and undergoes local unfolding reactions. This classified the unlocked state as a dry molten globule (DMG), which was proposed to represent an expanded native state with loosened side-chain interactions and a solvent-shielded core. To test whether the unlocked state of HP35 is actually expanded compared to the locked state, we performed high-pressure TTET measurements. Increasing pressure shifts the equilibrium from the locked toward the unlocked state, with a small negative reaction volume for unlocking (Δ V 0 = − 1.6 ± 0.5 cm 3 /mol). Therefore, rather than being expanded, the unlocked state represents an alternatively packed, compact state, demonstrating that native proteins can exist in several compact folded states, an observation with implications for protein function. The transition state for unlocking/locking, in contrast, has a largely increased volume relative to the locked and unlocked state, with respective activation volumes of 7.1 ± 0.4 cm 3 /mol and 8.7 ± 0.9 cm 3 /mol, indicating an expansion of the protein during the locking/unlocking transition. The presented results demonstrate the existence of both compact, low-energy and expanded, high-energy DMGs, prompting a broader definition of this state.

Volume changes associated with protein folding reactions contain valuable information about the folding mechanism and the nature of the transition state. However, meaningful interpretation of such data requires that overall volume changes be deconvoluted into individual contributions from different structural components. Here we focus on one type of structural element, the α-helix, and measure triplet-triplet energy transfer at high pressure to determine volume changes associated with the helix-coil transition. Our results reveal that the volume of a 21-amino-acid alanine-based peptide shrinks upon helix formation. Thus, helices, in contrast with native proteins, become more stable with increasing pressure, explaining the frequently observed helical structures in pressure-unfolded proteins. Both helix folding and unfolding become slower with increasing pressure. The volume changes associated with the addition of a single helical residue to a preexisting helix were obtained by comparing the experimental results with Monte Carlo simulations based on a kinetic linear Ising model. The reaction volume for adding a single residue to a helix is small and negative (-0.23 cm(3) per mol = -0.38 Å(3) per molecule) implying that intrahelical hydrogen bonds have a smaller volume than peptide-water hydrogen bonds. In contrast, the transition state has a larger volume than either the helical or the coil state, with activation volumes of 2.2 cm(3)/mol (3.7 Å(3) per molecule) for adding and 2.4 cm(3)/mol (4.0 Å(3) per molecule) for removing one residue. Thus, addition or removal of a helical residue proceeds through a transitory high-energy state with a large volume, possibly due to the presence of unsatisfied hydrogen bonds, although steric effects may also contribute.

The dynamics of peptide α-helices have been studied extensively for many years, and the kinetic mechanism of the helix–coil dynamics has been discussed controversially. Recent experimental results have suggested that equilibrium helix–coil dynamics are governed by movement of the helix/coil boundary along the peptide chain, which leads to slower unfolding kinetics in the helix center compared with the helix ends and position-independent helix formation kinetics. We tested this diffusion of boundary model in helical peptides of different lengths by triplet-triplet energy transfer measurements and compared the data with simulations based on a kinetic linear Ising model. The results show that boundary diffusion in helical peptides can be described by a classical, Einstein-type, 1D diffusion process with a diffusion coefficient of 2.7⋅10 7 (amino acids) 2 /s or 6.1⋅10 −9 cm 2 /s. In helices with a length longer than about 40 aa, helix unfolding by coil nucleation in a helical region occurs frequently in addition to boundary diffusion. Boundary diffusion is slowed down by helix-stabilizing capping motifs at the helix ends in agreement with predictions from the kinetic linear Ising model. We further tested local and nonlocal effects of amino acid replacements on helix–coil dynamics. Single amino acid replacements locally affect folding and unfolding dynamics with a ϕ f -value of 0.35, which shows that interactions leading to different helix propensities for different amino acids are already partially present in the transition state for helix formation. Nonlocal effects of amino acid replacements only influence helix unfolding ( ϕ f = 0) in agreement with a diffusing boundary mechanism.

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.

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.

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 α-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 μM and the concomitant detection of conformational transitions with rate constants of up to 8 s − 1 .

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.

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°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.

The C-terminal domain of T4 fibritin (foldon) is obligatory for the formation of the fibritin trimer structure and can be used as an artificial trimerization domain. Its native structure consists of a trimeric beta-hairpin propeller. At low pH, the foldon trimer disintegrates into a monomeric (A-state) form that has similar properties as that of an early intermediate of the trimer folding pathway. The formation of this A-state monomer from the trimer, its structure, thermodynamic stability, equilibrium association and folding dynamics have been characterized to atomic detail by modern high-resolution NMR techniques. The foldon A-state monomer forms a beta-hairpin with intact and stable H-bonds that is similar to the monomer in the foldon trimer, but lacks a defined structure in its N and C-terminal parts. Its thermodynamic stability in pure water is comparable to designed hairpins stabilized in alcohol/water mixtures. Details of the thermal unfolding of the foldon A-state have been characterized by chemical shifts and residual dipolar couplings (RDCs) detected in inert, mechanically stretched polyacrylamide gels. At the onset of the thermal transition, uniform relative changes in RDC values indicate a uniform decrease of local N-HN and Calpha-Halpha order parameters for the hairpin strand residues. In contrast, near-turn residues show particular thermal stability in RDC values and hence in local order parameters. This coincides with increased transition temperatures of the beta-turn residues observed by chemical shifts. At high temperatures, the RDCs converge to non-zero average values consistent with predictions from random chain polymer models. Residue-specific deviations above the unfolding transition reveal the persistence of residual order around proline residues, large hydrophobic residues and at the beta-turn.

The capsid protein of Semliki Forest virus constitutes the N-terminal part of a large viral polyprotein. It consists of an unstructured basic segment (residues 1-118) and a 149 residue serine protease module (SFVP, residues 119-267) comprised of two beta-barrel domains. Previous in vivo and in vitro translation experiments have demonstrated that SFVP folds co-translationally during synthesis of the viral polyprotein and rapidly cleaves itself off the nascent chain. To test whether fast co-translation folding of SFVP is an intrinsic property of the polypeptide chain or whether folding is accelerated by cellular components, we investigated spontaneous folding of recombinant SFVP in vitro. The results show that the majority of unfolded SFVP molecules fold faster than any previously studied two-domain protein (tau=50 ms), and that folding of the N-terminal domain precedes structure formation of the C-terminal domain. This shows that co-translational folding of SFVP does not require additional cellular components and suggests that rapid folding is the result of molecular evolution towards efficient virus biogenesis.

The rate of formation of intramolecular interactions in unfolded proteins determines how fast conformational space can be explored during folding. Characterization of the dynamics of unfolded proteins is therefore essential for the understanding of the earliest steps in protein folding. We used triplet-triplet energy transfer to measure formation of intrachain contacts in different unfolded polypeptide chains. The time constants (1/k) for contact formation over short distances are almost independent of chain length, with a maximum value of about 5ns for flexible glycine-rich chains and of 12ns for stiffer chains. The rates of contact formation over longer distances decrease with increasing chain length, indicating different rate-limiting steps for motions over short and long chain segments. The effect of the amino acid sequence on local chain dynamics was probed by using a series of host-guest peptides. Formation of local contacts is only sixfold slower around the stiffest amino acid (proline) compared to the most flexible amino acid (glycine). Good solvents for polypeptide chains like EtOH, GdmCl and urea were found to slow intrachain diffusion and to decrease chain stiffness. These data allow us to determine the time constants for formation of the earliest intrachain contacts during protein folding. © 2003 Elsevier Ltd. All rights reserved.

Apparent transition state movement upon mutation or changes in solvent conditions is frequently observed in protein folding and is often interpreted in terms of Hammond behavior. This led to the conclusion that barrier regions in protein folding are broad maxima on the free energy landscape. Here, we use the concept of self-interaction and cross-interaction parameters to test experimental data of 21 well-characterized proteins for Hammond behavior. This allows us to characterize the origin of transition state movements along different reaction coordinates. Only one of the 21 proteins shows a small but coherent transition state movement in agreement with the Hammond postulate. In most proteins the structure of the transition state is insensitive to changes in protein stability. The apparent change in the position of the transition state upon mutation, which is frequently observed in phi-value analysis, is in most cases due to ground-state effects caused by structural changes in the unfolded state. This argues for significant residual structure in unfolded polypeptide chains of many proteins. Disruption of these residual interactions by mutation often leads to decreased folding rates, which implies that these interactions are still present in the transition state. The failure to detect Hammond behavior shows that the free energy barriers encountered by a folding polypeptide chain are generally rather narrow and robust maxima for all experimentally explorable reaction coordinates.

Many small proteins fold fast and without detectable intermediates. This is frequently taken as evidence against the importance of partially folded states, which often transiently accumulate during folding of larger proteins. To get insight into the properties of free energy barriers in protein folding we analyzed experimental data from 23 proteins that were reported to show non-linear activation free-energy relationships. These non-linearities are generally interpreted in terms of broad transition barrier regions with a large number of energetically similar states. Our results argue against the presence of a single broad barrier region. They rather indicate that the non-linearities are caused by sequential folding pathways with consecutive distinct barriers and a few obligatory high-energy intermediates. In contrast to a broad barrier model the sequential model gives a consistent picture of the folding barriers for different variants of the same protein and when folding of a single protein is analyzed under different solvent conditions. The sequential model is also able to explain changes from linear to non-linear free energy relationships and from apparent two-state folding to folding through populated intermediates upon single point mutations or changes in the experimental conditions. These results suggest that the apparent discrepancy between two-state and multi-state folding originates in the relative stability of the intermediates, which argues for the importance of partially folded states in protein folding.

During the folding of many proteins, collapsed globular states are formed prior to the native structure. The role of these states for the folding process has been widely discussed. Comparison with properties of synthetic homo and heteropolymers had suggested that the initial collapse represented a shift of the ensemble of unfolded conformations to more compact states without major energy barriers. We investigated the folding/unfolding transition of a collapsed state, which transiently populates early in lysozyme folding. This state forms within the dead-time of stopped-flow mixing and it has been shown to be significantly more compact and globular than the denaturant-induced unfolded state. We used the GdmCl-dependence of the dead-time signal change to characterize the unfolding transition of the burst phase intermediate. Fluorescence and far-UV CD give identical unfolding curves, arguing for a cooperative two-state folding/unfolding transition between unfolded and collapsed lysozyme. These results show that collapse leads to a distinct state in the folding process, which is separated from the ensemble of unfolded molecules by a significant energy barrier. NMR, fluorescence and small angle X-ray scattering data further show that some local interactions in unfolded lysozyme exist at denaturant concentrations above the coil-collapse transition. These interactions might play a crucial role in the kinetic partitioning between fast and slow folding pathways.

Folding of tendamistat, an inhibitor of α-amylase, is a fast two-state process accompanied by two minor slow reactions, which were assigned to prolyl isomerization. In a proline-free variant, 5% of the molecules still fold slowly with a rate constant of 2.5 s−1. This reaction is caused by a slow equilibrium between two populations of unfolded molecules. The time constant for this equilibration process, its sensitivity to LiCl and its temperature dependence identify it as a cis-trans isomerization of nonprolyl peptide bonds. Although nonprolyl peptide bonds have the cis conformation populating only ∼0.15% in unfolded proteins, their large number generates a significant fraction of slow-folding molecules. This emphasizes that heterogeneous populations in an unfolded protein can induce complex folding kinetics on various time scales.

The small all-β-sheet protein tendamistat folds and unfolds rapidly in apparent two-state reactions. Kinetic measurements of two tendamistat variants under various solvent conditions reveal, however, that folding occurs in at least two sequential steps through a metastable obligatory intermediate. Depending on the solvent conditions either step can become rate limiting. The activation parameters indicate that the first step represents an enthalpic barrier whereas the second step is an entropic barrier at 25 °C. Our results suggest that initial non-specific collapse precedes formation of native secondary and tertiary structure in tendamistat folding. This points at a distinct route in tendamistat folding and indicates that partially folded metastable intermediates might play an important role in the mechanism of apparent two-state folding.

Lysozyme folds through two competing pathways. A fast pathway leads directly from a collapsed state to the native protein, whereas folding on a slow pathway proceeds through a partially folded intermediate (I(1)). At NaCl concentrations above 100 mM, a second transient intermediate (I(2)) is induced as judged by the appearance of an additional apparent rate constant in the refolding kinetics. Monitoring the time course of native molecules and of both intermediates shows that the NaCl-induced state (I(2)) is located on neither of the two folding pathways observed at low-salt concentrations. These results suggest that I(2) is a metastable high-energy intermediate at low-ionic strength and is located on a third folding pathway. The folding landscape of lysozyme seems to be complex with several high-energy intermediates located on parallel folding routes. However, the experiments show no evidence for partially folded states on the fast direct pathway.

A direct measure of intramolecular chain diffusion is obtained by the determination of triplet–triplet energy-transfer rates between a donor and an acceptor chromophore attached at defined points on a polypeptide chain. Single exponential kinetics of contact formation are observed on the nanosecond time scale for polypeptides in which donor and acceptor are linked by repeating units of glycine and serine residues. The rates depend on the number of peptide bonds ( N ) separating donor and acceptor and show a maximum for the shortest peptides ( N = 3) with a time constant ( τ = 1/ k ) of 20 ns. This sets an upper limit for the speed of formation of the first side-chain contacts during protein folding.

The effect of intermediates on the rate of protein folding is explored by applying Kramers’ theory of diffusive barrier crossing in the high friction limit. Intermediates are represented as local minima in the transition barrier. We observe that very large or very small additional barriers created by the intermediates slow down the folding process. The rate of folding markedly increases, however, when the additional barriers become >1 k B T but leave the overall barrier height unchanged. This rate-enhancing effect is caused by a favorable entropic contribution to the free energy of activation, and it increases with the number of intermediates up to a limiting value. From these calculations, we conclude that optimized transition barriers should contain partially folded high energy intermediates.

Wie ahnlich sind Faltungsintermediate dem nativen Protein, und welches ist der geschwindigkeitsbestimmende Schritt bei der Proteinfaltung? Unterstutzen neue Ribozyme die RNA-Welt-Hypothese? In der Genomforschung ist die Halfte der Zeit um - erreichte man auch die Halfte der angestrebten Ziele? Und welche Datenbank informiert den Suchenden uber den Stand der Genomforschung? Die diesjahrigen Trendberichte gehen diesen Fragen nach.

We investigated the reversible folding and unfolding reactions of the small 74 amino acid residue protein tendamistat. The secondary structure of tendamistat contains only β-sheets and loop regions and the protein contains two disulfide bonds. Fluorescence-detected refolding kinetics of tendamistat (disulfide bonds intact) comprise of a major rapid fast reaction (τ=10 ms in water) and two minor slow reactions. In the fast reaction 80% of the unfolded molecules are converted to native protein. The two slow reactions are part of a parallel slow folding pathway. On this pathway the rate-limiting step in the formation of native molecules is cis to trans isomerization of at least one of the three trans Xaa-Pro peptide bonds. This reaction is catalyzed efficiently by the enzyme peptidyl-prolyl cis-trans isomerase. Comparison of kinetic data with equilibrium unfolding transitions shows that the fast folding pathway follows a two-state process without populated intermediate states. Additionally, various sensitive tests did not detect any rapid chain collapse during tendamistat folding prior to the acquisition of the native three-dimensional structure. These results show that pre-formed disulfide bonds do not prevent efficient and rapid protein folding.

A kinetic folding intermediate of hen lysozyme is shown to form in a nucleation/growth type of mechanism. Under native solvent conditions, a nucleated state is formed slowly during refolding (tau = 14 +/- 1 ms at 0 M GdmCl) and is rapidly converted to the folding intermediate (tau = 300 +/- 150 micros at 0 M GdmCl). Under these conditions the nucleated state represents a high-energy state compared to the folding intermediate (delta deltaG0 = 13.7 +/- 3 kJ/mol). At elevated concentrations of GdmCl, the nucleated state becomes more stable than the intermediate and it consequently becomes transiently populated during unfolding of the intermediate state. This allowed us to measure the rate constant of the growth step using stopped-flow double-jump experiments. At high concentrations of GdmCl (>5 M), the growth step becomes rate-limiting in unfolding, leading to the frequently observed rollover in the GdmCl dependence of the logarithm of the apparent rate constant of the unfolding reaction.

The effect of trifluoroethanol (TFE) on the structure of the all-beta-sheet protein tendamistat was investigated. At low concentrations TFE induces cooperative loss of the native tertiary structure leading to a partially folded state. The loss of specific side-chain interactions in the transition from the native state of the TFE-induced state is demonstrated by the disappearance of the CD bands in the aromatic region, a reduced chemical shift dispersion of the one-dimensional 1H NMR spectrum and a broad, uncooperative thermal unfolding transition of the partially folded state. An increased line-width of the NMR bands in the TFE state compared with the unfolded state suggests the presence of multiple, rapidly interconverting conformations. Hydrogen-exchange studies of amide proteins in the TFE state reveal the existence of defined hydrogen bonds at the same locations as in the native state, but with largely reduced stability. This suggests the presence of most of the native beta-sheet structure. These results are supported by Fourier transformed IR measurements, which show nearly the same amount of beta-structure in the TFE state and in the native state. Far UV CD spectroscopy suggests the induction of some alpha-helical structure upon addition of TFE, which appears to be located mainly in regions corresponding to loops or random structure in the native state and which seems to represent fluctuating conformations with preferred backbone angles rather than stable, hydrogen-bonded alpha-helices. These results show that stable non-local interactions, as they occur in beta-sheets, can form in the absence of specific side-chain interactions. The presence of a subset of the native long-range interactions and the absence of stable non-native interactions suggests that the observed partially folded state might represent an early intermediate on a hierarchical folding pathway of tendamistat.

Recently, when the kinetic unfolding process of ribonuclease A was monitored by hydrogen exchange (T. Kiefhaber and R.L. Baldwin, Proc. Natl. Acad. Sci. USA, 92 (1995) 2657–2661), all peptide hydrogen bonds were found to undergo rapid exchange in a single kinetic step under conditions where unfolding is slow and the intrinsic rate of hydrogen exchange is fast (pH 8.0, 10°C, 4.5 M guanidinium chloride). Comparison with the unfolding rate measured by circular dichroism indicates that hydrogen exchange is caused by the rate-limiting step of unfolding. No evidence was found for partly unfolded intermediates that are formed slowly enough to be observed by EX1 (unfolding-limited) hydrogen exchange. Some peptide NH protons were found to show, in addition to EX1 exchange, faster EX2 exchange that is base-catalyzed. The EX2 exchange is caused by species that equilibrate rapidly with the native protein at the start of the unfolding process. These species might include rapidly formed unfolding intermediates. We show here that any such unfolding intermediates must have large protection factors because the EX2 reactions of ribonuclease A under these unfolding conditions have protection factors ≥ 2500.

When NMR hydrogen exchange was used previously to monitor the kinetics of RNase A unfolding, some peptide NH protons were found to show EX2 exchange (detected by base catalysis) in addition to the expected EX1 exchange, whose rate is limited by the kinetic unfolding process. In earlier work, two groups showed independently that a restricted two-process model successfully fits published hydrogen exchange rates of native RNase A in the range 0-0.7 M guanidinium chloride. We find that this model predicts properties that are very different from the observed properties of the EX2 exchange reactions of RNase A in conditions where guanidine-induced unfolding takes place. The model predicts that EX2 exchange should be too fast to measure by the technique used, whereas it is readily measurable. Possible explanations for the contradiction are considered here, and we show that removing the restriction from the earlier two-process model is sufficient to resolve the contradiction; instead of specifying that exchange caused by global unfolding occurs by the EX2 mechanism, we allow it to occur by the general mechanism, which includes both the EX1 and EX2 cases. It is logical to remove this restriction because global unfolding of RNase A is known to give rise to EX1 exchange in these unfolding conditions. Resolving the contradiction makes it possible to determine whether populated unfolding intermediates contribute to the EX2 exchange, and this question is considered elsewhere. The results and simulations indicate that moderate or high denaturant concentrations readily give rise to EX1 exchange in native proteins. Earlier studies showed that hydrogen exchange in native proteins typically occurs by the EX2 mechanism but that high temperatures or pH values above 7 may give rise to EX1 exchange. High denaturant concentrations should be added to the list of variables likely to cause EX1 exchange.

Die 3D-Strukturen von Cytochrom c-Oxidase und Proteasom wurden atomar aufgelost, das Wissen uber Tubulin, Ribosom und Spleisosom erweitert. Peptidmodelle und die Charakterisierung teilgefalteter Strukturen helfen, die Proteinfaltung aufzuklaren. Gen- “Targeting” beschleunigt die Neurorezeptorforschung. An Beispielen warden Fortschritte der Molekularen Medizin beschrieben. Biologische Systeme warden nicht mehr nur “genutzt”, sondern durch Biotechnologie “nutzbar gemacht”.

Folding of lysozyme from hen egg white was investigated by using interrupted refolding experiments. This method makes use of a high energy barrier between the native state and transient folding intermediates, and, in contrast to conventional optical techniques, it enables one to specifically monitor the amount of native molecules during protein folding. The results show that under strongly native conditions lysozyme can refold on parallel pathways. The major part of the lysozyme molecules (86%) refold on a slow kinetic pathway with well-populated partially folded states. Additionally, 14% of the molecules fold faster. The rate constant of formation of native molecules on the fast pathway corresponds well to the rate constant expected for folding to occur by a two-state process without any detectable intermediates. The results suggest that formation of the native state for the major fraction of lysozyme molecules is retarded compared with the direct folding process. Partially structured intermediates that transiently populate seem to be kinetically trapped in a conformation that can only slowly reach the native structure.

During acid-induced unfolding of apomyoglobin, a partly folded form is observed at pH values of around four. In this form, the A, G and H helices are folded, while the rest of the molecule, including the B helix, demonstrates little structure. The partly folded form has been described as a molten globule form. To determine the factors that govern the structure and stability of this form, we introduced two helix-stabilizing mutations into the B helix, and tested their effect on the structure and stability of both the native form and the molten globule form. We show that the two Gly→Ala replacements in the B helix produce altered flluorescence and CD properties of the partly folded intermediate, a result which implies that the B helix has become part of the structured region of the molten globule form. The helix content of a model peptide containing the sequence of the B helix is increased by the G→A replacements, as is the helix content of the molten globule intermediate, whereas the stability and the helix content of the native protein are not altered. The observed increase in helicity is larger in the folding intermediate than in the model peptide, suggesting that nonspecific interactions, such as the hydrophobic interactions exhibited by the entire polypeptide chain, amplify the effect of intrinsic helix stability. The overall results suggest that the intrinsic stability of each individual helix is a factor in deciding whether or not that helix becomes part of the structured molten globule.

The intramolecular and intermolecular vibrational energy flow in a polyproline peptide with a total number of nine amino acids in the solvent dimethyl sulfoxide is investigated using time-resolved infrared (IR) spectroscopy. Azobenzene covalently bound to a proline sequence containing nitrophenylalanine as a local sensor for vibrational excess energy serves as a heat source. Information on through-space distances in the polyproline peptides is obtained by independent Forster resonance energy transfer measurements. Photoexcitation of the azobenzene and subsequent internal conversion yield strong vibrational excitation of the molecule acting as a local heat source. The relaxation of excess heat, its transfer along the peptide and to the solvent is monitored by the response of the nitro-group in nitrophenylalanine acting as internal thermometer. After optical excitation, vibrational excess energy is observed via changes in the IR absorption spectra of the peptide. The nitrophenylalanine bands reveal that the vibrational excess energy flows in the peptide over distances of more than 20 A and arrives delayed by up to 7 ps at the outer positions of the peptide. The vibrational excess energy is transferred to the surrounding solvent on a time scale of 10-20 ps. The experimental observations are analyzed by different heat conduction models. Isotropic heat conduction in three dimensions away from the azobenzene heat source is not able to describe the observations. One-dimensional heat dissipation along the polyproline peptide combined with a slower transversal heat transfer to the solvent surrounding well reproduces the observations.

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.

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.

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 (Δ H 0 = 35 ± 4 kJ/mol) which is nearly compensated in free energy by the entropy change (Δ S 0 = 112 ± 20 J·mol -1 ·K -1 ). The unlocking reaction has a time constant of about 1 μs at 5 °C and is enthalpy-limited with an activation energy of 32 ± 1 kJ/mol and a large Arrhenius preexponential factor of A = 7.5 × 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.

Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single alpha-helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the beta-sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (tau = 6,500 s at pH 5.0 and 10 degrees C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties.

Thermal and GdmCl-induced unfolding transitions of aldolase from Staphylococcus aureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a delta G0 value of -9 +/- 2 kJ/mol at 20 degrees C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 degrees C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of alpha/beta barrel proteins.

The folding and unfolding kinetics within the transition region were measured for RNase A and for RNase T1. The data were used to evaluate the theoretical models for the influence of prolyl isomerization on the observed folding kinetics. These two proteins were selected, since the folding reaction of RNase A is faster than prolyl isomerization, whereas in RNase T1, folding is slower than isomerization in the transition region. Folding of RNase T1 was investigated for three variants with different numbers of cis prolyl residues. The results indicate that in the transition region the folding rates are indeed strongly dependent on the number of prolyl residues. The variant of RNase T1 that contains only one cis prolyl residue folds about ten times faster than two variants that contain two cis prolyl residues. For both RNase A and RNase T1, the apparent rates of folding and unfolding as well as the corresponding amplitudes depend on the concentration of denaturant in a manner that was predicted by the model calculations. When refolding was started from the fast-folding species, additional kinetic phases could be observed in the transition region for both proteins. The obtained values could be used to calculate the microscopic rate constants of folding and isomerization on the basis of theoretical models.

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.

Protein aggregation is frequently observed as a major side-reaction of protein folding. We present quantitative models explaining the formation of aggregates during protein folding in vitro and in vivo on the basis of a kinetic competition between correct folding and aggregation reactions. Both models are in good agreement with experimental data. The model implies that, in vitro, the yield of native protein obtained upon refolding is determined by the rates of the competing first order folding and second order aggregation reactions. Therefore, a high protein concentrations aggregation dominates over folding and leads to the formation of insoluble protein. For in vivo protein synthesis, the model shows that the yield of native protein is only dependent on the rate of folding, on the rate of aggregation and on the rate of protein synthesis. In the cell, several mechanisms, including "folding helpers" seem to have evolved, which influence these processes and thereby prevent unproductive side reactions.

Originally published in: Protein Folding Handbook. Part I. Edited by Johannes Buchner and Thomas Kiefhaber. Copyright © 2005 Wiley-VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3-527-30784-2 The sections in this article are Introduction Analysis of Protein Folding Reactions using Simple Kinetic Models General Treatment of Kinetic Data Two-state Protein Folding Complex Folding Kinetics Heterogeneity in the Unfolded State Folding through Intermediates Rapid Pre-equilibria Folding through an On-pathway High-energy Intermediate A Case Study: the Mechanism of Lysozyme Folding Lysozyme Folding at pH 5.2 and Low Salt Concentrations Lysozyme Folding at pH 9.2 or at High Salt Concentrations Non-exponential Kinetics Conclusions and Outlook Protocols–Analytical Solutions of Three-state Protein Folding Models Triangular Mechanism On-pathway Intermediate Off-pathway Mechanism Folding Through an On-pathway High-Energy Intermediate Acknowledgments Keywords: protein folding; two-state folding; folding intermediates; lysozyme; folding pathways

It is our aim to elucidate molecular aspects of the mechanism of protein folding. We use ribonuclease T1 as a model protein, because it is a small single-domain protein with a well-defined secondary and tertiary structure, which is stable in the presence and absence of disulfide bonds. Also, an efficient mutagenesis system is available to produce protein molecules with defined sequence variations. Here we present a preliminary characterization of the folding kinetics of ribonuclease T1. Its unfolding and refolding reactions are reversible, which is shown by the quantitative recovery of the catalytic activity after an unfolding/refolding cycle. Refolding is a complex process, where native protein is formed on three distinguishable pathways. There are 3.5% fast-folding molecules, which refold within the millisecond time range, and 96.5% slow-folding species, which regain the native state in the time range of minutes to hours. These slow-folding molecules give rise to two major, parallel refolding reactions. The mixture of fast- and slow-folding molecules is produced slowly after unfolding by chain equilibration reactions that show properties of proline isomerization. We conclude that part of the kinetic complexity of RNase T1 folding can be explained on the basis of the proline model for protein folding. This is supported by the finding that the slow refolding reactions of this protein are accelerated in the presence of the enzyme prolyl isomerase. However, several properties of ribonuclease T1 refolding, such as the dependence of the relative amplitudes on the probes, used to follow folding, are not readily explained by a simple proline model.

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.

Intersystem crossing of xanthone in protic solvents proceeds within ∼ 1 ps, whereas its fluorescence decays within 60–600 ps. This is due to a delayed fluorescence from a 3 nπ* state energetically close to the 1 ππ* state.

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.

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 Å 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) × 10 −7 cm 2 s −1 in water to (14.8 ± 2.5) × 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.

Photo-excited xanthone is known to undergo ultrafast intersystem crossing (ISC) in the 1 ps time domain. Correspondingly, its fluorescence quantum yield in most solvents is very small ( approximately 10(-4)). Surprisingly, the quantum yield in water is 100 times larger, while ISC is still rapid ( approximately 1 ps), as seen by ultrafast pump probe absorption spectroscopy. Temperature dependent steady state and time resolved fluorescence experiments point to a delayed fluorescence mechanism, where the triplet (3)npi* state primarily accessed by ISC is nearly isoenergetic with the photo-excited (1)pipi* state. The delayed fluorescence of xanthone in water decays with a time constant of 700 ps, apparently by internal conversion between the (3)npi* state and the lowest lying triplet state (3)pipi*.

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.

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.

PART I: Principles of Protein Folding and Stability. Spectroscopic techniques to study protein folding and stability. Solvent effects on protein stability. Analysis of protein folding kinetics, kinetic mechanisms, transition states. Single molecule protein folding. Dynamics of unfolded polypeptide chains. Folding and stability of oligomeric proteins. Folding of membrane proteins. Molecular dynamics simulations of folding and unfolding. Ab initio methods for protein structure prediction. PART II: Protein Misfolding, Molecular Chaperones and Folding Catalysts. Methods to study aggregate formation. Amyloid diseases. Polyglutamine repeat diseases. Protein refolding technology. Engineering protein stability. Chaperone machines. Folding of nascent polypeptides. Protein assembly processes.

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.