Relationship between the native-state hydrogen exchange and folding pathways of a four-helix bundle protein

The hydrogen exchange behavior of a four-helix bundle protein in low concentrations of denaturant reveals some partially unfolded forms that are significantly more stable than the fully unfolded state. Kinetic folding of the protein, however, is apparently two-state in the absence of the accumulation of early folding intermediates. The partially unfolded forms are either as folded as or more folded than the rate-limiting transition state and appear to represent the major intermediates in a folding and unfolding reaction. These results are consistent with the suggestion that partially unfolded intermediates may form after the rate-limiting step for small proteins with apparent two-state folding kinetics.     

Sensitivity of NMR residual dipolar couplings to perturbations in folded and denatured staphylococcal nuclease

The invariance of NMR residual dipolar couplings (RDCs) in denatured forms of staphylococcal nuclease to changes in denaturant concentration or amino acid sequence has previously been attributed to the robustness of long-range structure in the denatured state. Here we compare RDCs of the wild-type nuclease with those of a fragment that retains a folded OB-fold subdomain structure despite missing the last 47 of 149 residues. The RDCs of the intact protein and of the truncation fragment are substantially different under conditions that favor folded structure. By contrast, there is a strong correlation between the RDCs of the full-length protein and the fragment under denaturing conditions (6 M urea). The RDCs of the folded and unfolded forms of the proteins are uncorrelated. Our results suggest that RDCs are more sensitive to structural changes in folded than unfolded proteins. We propose that the greater susceptibility of RDCs in folded states is a consequence of the close packing of the polypeptide chain under native conditions. By contrast, the invariance of RDCs in denatured states is more consistent with a disruption of cooperative structure than with the retention of a unique long-range folding topology.     

Single-molecule FRET study of denaturant induced unfolding of RNase H

Single-molecule fluorescence (F?rster) resonance energy transfer (FRET) experiments were performed on surface-immobilized RNase H molecules as a function of the concentration of the chemical denaturant guanidinium chloride (GdmCl). For comparison, we measured ensemble FRET on RNase H solutions. The single-molecule approach allowed us to study FRET distributions of the subpopulation of unfolded molecules without interference from the folded population. The unfolded ensemble experienced a continuous shift of the FRET efficiency distribution with increasing concentration of GdmCl, indicating a heterogeneous population of expanding, unfolded polypeptide chains. We have analyzed the behavior of the unfolded state quantitatively with a model in which the unfolded state is described by a continuum of substates, with the free energy of each substate linearly coupled to its m-value, the proportionality coefficient between free energy and denaturant activity. By fitting this model to the data, we have derived energetic and structural parameters that describe the unfolded state ensemble. Specifically, we have found that the average size of the unfolded state increases from 23-38 A between 0 and 6 M denaturant. Excellent agreement was achieved between the fitted model and our FRET measurements, and with previously published nuclear magnetic resonance and small-angle X-ray scattering data.     

Specificity of the initial collapse in the folding of the cold shock protein

The two-state folding reaction of the cold shock protein from Bacillus caldolyticus (Bc-Csp) is preceded by a rapid chain collapse. A fast shortening of intra-protein distances was revealed by F?rster resonance energy transfer (FRET) measurements with protein variants that carried individual pairs of donor and acceptor chromophores at various positions along the polypeptide chain. Here we investigated the specificity of this rapid compaction. Energy transfer experiments that probed the stretching of strand beta2 and the close approach between the strands beta1 and beta2 revealed that the beta1-beta2 hairpin is barely formed in the collapsed form, although it is native-like in the folding transition state of Bc-Csp. The time course of the collapse could not be resolved by pressure or temperature jump experiments, indicating that the collapsed and extended forms are not separated by an energy barrier. The co-solute (NH4)2SO4 stabilizes both native Bc-Csp and the collapsed form, which suggests that the large hydrated SO4(2-) ions are excluded from the surface of the collapsed form in a similar fashion as they are excluded from folded Bc-Csp. Ethylene glycol increases the stability of proteins because it is excluded preferentially from the backbone, which is accessible in the unfolded state. The collapsed form of Bc-Csp resembles the unfolded form in its interaction with ethylene glycol, suggesting that in the collapsed form the backbone is still accessible to water and small molecules. Our results thus rule out that the collapsed form is a folding intermediate with native-like chain topology. It is better described as a mixture of compact conformations that belong to the unfolded state ensemble. However, some of its structural elements are reminiscent of the native protein.     

Nonequilibrium single molecule protein folding in a coaxial mixer

We have developed a continuous-flow mixing device suitable for monitoring bioconformational reactions at the single-molecule level with a response time of ∼10 ms under single-molecule flow conditions. Its coaxial geometry allows three-dimensional hydrodynamic focusing of sample fluids to diffraction-limited dimensions where diffusional mixing is rapid and efficient. The capillary-based design enables rapid in-lab construction of mixers without the need for expensive lithography-based microfabrication facilities. In-line filtering of sample fluids using granulated silica particles virtually eliminates clogging and extends the lifetime of each device to many months. In this article, to determine both the distance-to-time transfer function and the instrument response function of the device we characterize its fluid flow and mixing properties using both fluorescence cross-correlation spectroscopy velocimetry and finite element fluid dynamics simulations. We then apply the mixer to single molecule FRET protein folding studies of Chymotrypsin Inhibitor protein 2. By transiently populating the unfolded state of Chymotrypsin Inhibitor Protein 2 (CI2) under nonequilibrium in vitro refolding conditions, we spatially and temporally resolve the denaturant-dependent nonspecific collapse of the unfolded state from the barrier-limited folding transition of CI2. Our results are consistent with previous CI2 mixing results that found evidence for a heterogeneous unfolded state consisting of cis- and trans-proline conformers.     

Thermodynamics and Kinetics of Single-Chain Monellin Folding with Structural Insights into Specific Collapse in the Denatured State Ensemble

Proteins, which behave as random coils in high denaturant concentrations undergo collapse transition similar to polymers on denaturant dilution. We study collapse in the denatured ensemble of single-chain monellin (MNEI) using a coarse-grained protein model and molecular dynamics simulations. The model is validated by quantitatively comparing the computed guanidinium chloride and pH-dependent thermodynamic properties of MNEI folding with the experiments. The computed properties such as the fraction of the protein in the folded state and radius of gyration (R_g) as function of [GuHCl] are in good agreement with the experiments. The folded state of MNEI is destabilized with an increase in pH due to the deprotonation of the residues Glu24 and Cys42. On decreasing [GuHCl], the protein in the unfolded ensemble showed specific compaction. The R_g of the protein decreased steadily with [GuHCl] dilution due to increase in the number of native contacts in all the secondary structural elements present in the protein. MNEI folding kinetics is complex with multiple folding pathways and transiently stable intermediates are populated in these pathways. In strong stabilizing conditions, the protein in the unfolded ensemble showed transition to a more compact unfolded state where R_g decreased by ≈ 17% due to the formation of specific native contacts in the protein. The intermediate populated in the dominant MNEI folding pathway satisfies the structural features of the dry molten globule inferred from experiments.     

Characterization of the hydrodynamic properties of the folding transition state of an SH3 domain by magnetization transfer NMR spectroscopy

Protein folding kinetic data have been obtained for the marginally stable N-terminal Src homology 3 domain of the Drosophila protein drk (drkN SH3) in an investigation of the hydrodynamic properties of its folding transition state. Due to the presence of NMR resonances of both folded and unfolded states at equilibrium, kinetic data can be derived from NMR magnetization transfer techniques under equilibrium conditions. Kinetic analysis as a function of urea (less than approximately 1 M) and glycerol enables determination of alpha values, measures of the energetic sensitivity of the transition state to the perturbation relative to the end states of the protein folding reaction (the folded and unfolded states). Both end states have previously been studied experimentally by NMR spectroscopic and other biophysical methods in great detail and under nondenaturing conditions. Combining these results with the kinetic folding data obtained here, we can characterize the folding transition state without requiring empirical models for the unfolded state structure. We are thus able to give a reliable measure of the solvent-accessible surface area of the transition state of the drkN SH3 domain (4730 +/- 360 A(2)) based on urea titration data. Glycerol titration data give similar results and additionally demonstrate that folding of this SH3 domain is dependent on solvent viscosity, which is indicative of at least partial hydration of the transition state. Because SH3 domains appear to fold by a common folding mechanism, the data presented here provide valuable insight into the transition states of the drkN and other SH3 domains.     

Molecular basis of cooperativity in protein folding IV. Core: A general cooperative folding model

The cooperative nature of the protein folding process is independent of the characteristic fold and the specific secondary structure attributes of a globular protein. A general folding/unfolding model should, therefore, be based upon structural features that transcend the peculiarities of α-helices, β-sheets, and other structural motifs found in proteins. The studies presented in this paper suggest that a single structural characteristic common to all globular proteins is essential for cooperative folding. The formation of a partly folded state from the native state results in the exposure to solvent of two distinct regions: (1) the portions of the protein that are unfolded; and (2) the “complementary surfaces,” located in the regions of the protein that remain folded. The cooperative character of the folding/unfolding transition is determined largely by the energetics of exposing complementary surface regions to the solvent. By definition, complementary regions are present only in partly folded states; they are absent from the native and unfolded states. An unfavorable free energy lowers the probability of partly folded states and increases the cooperativity of the transition. In this paper we present a mathematical formulation of this behavior and develop a general cooperative folding/unfolding model, termed the “complementary region” (CORE) model. This model successfully reproduces the main properties of folding/unfolding transitions without limiting the number of partly folded states accessible to the protein, thereby permitting a systematic examination of the structural and solvent conditions under which intermediates become populated. It is shown that the CORE model predicts two-state folding/unfolding behavior, even though the two-state character is not assumed in the model. © 1993 Wiley-Liss, Inc.     

Is it always possible to distinguish two- and three-state systems by evaluating the van't Hoff enthalpy?

Many small globular proteins are traditionally classified as thermodynamical two-state systems, i.e., the protein is either in the native, active state (folded) or in the denatured state (unfolded). We challenge this view and show that there may exist (protein) systems for which a van't Hoff analysis of experimental data cannot determine whether the system corresponds to two or three thermodynamical states when only temperatures in a narrow temperature region around the transition are considered. We generalize a widely employed two-state protein folding model to include a third, transition state. For this three-state system we systematically study the deviation of the calorimetric enthalpy (heat of transition) from the van't Hoff enthalpy, a measure of the two-stateness of a transition. We show that under certain conditions the heat capacity of the three-state system can be almost indistinguishable from the heat capacity for the two-state system over a broad temperature interval. The consequence may be that some three-state (or even more than three-states) systems have been misinterpreted as two-state systems when the conclusion is drawn solely upon the van't Hoff enthalpy. These findings are important not only for proteins, but also for the interpretation of thermodynamical systems in general.     

Test for cooperativity in the early kinetic intermediate in lysozyme 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.     

Natively unfolded human prothymosin alpha adopts partially folded collapsed conformation at acidic pH.

Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.     

Fast collapse but slow formation of secondary structure elements in the refolding transition of E. coli adenylate kinase

The various models proposed for protein folding transition differ in their order of appearance of the basic steps during this process. In this study, steady state and time-resolved dynamic non-radiative excitation energy transfer (FRET and trFRET) combined with site specific labeling experiments were applied in order to characterize the initial transient ensemble of Escherichia coli adenylate kinase (AK) molecules upon shifting conditions from those favoring denaturation to refolding and from folding to denaturing. Three sets of labeled AK mutants were prepared, which were designed to probe the equilibrium and transient distributions of intramolecular segmental end-to-end distances. A 176 residue section (residues 28-203), which spans most of the 214 residue molecule, and two short secondary structure chain segments including an alpha-helix (residues 169-188) and a predominantly beta-strand region (residues 188-203), were labeled. Upon fast change of conditions from denaturing to folding, the end-to-end distance of the 176 residue chain section showed an immediate collapse to a mean value of 26 A. Under the same conditions, the two short secondary structure elements did not respond to this shift within the first ten milliseconds, and retained the characteristics of a fully unfolded state. Within the first 10 ms after changes of the solvent from folding to denaturing, only minor changes were observed at the local environments of residues 203 and 169. The response of these same local environments to the shift of conditions from denaturing to folding occurred within the dead time of the mixing device. Thus, the response of the CORE domain of AK to fast transfer from folding to unfolding conditions is slow at all three conformational levels that were probed, and for at least a few milliseconds the ensemble of folded molecules is maintained under unfolding conditions. A different order of the changes was observed upon initiation of refolding. The AK molecules undergo fast collapse to an ensemble of compact structures where the local environment of surface probes seems to be native-like but the two labeled secondary structure elements remain unfolded.     

Pressure-jump studies of the folding/unfolding of trp repressor.

The dimeric protein, trp apo-repressor of Escherichia coli has been subjected to high hydrostatic pressure under a variety of conditions, and the effects have been monitored by fluorescence spectroscopic and infra-red absorption techniques. Under conditions of micromolar protein concentration and low, non-denaturing concentrations of guanidinium hydrochloride (GuHCl), tryptophan and 8-anilino-1-naphthalene sulfonate (ANS) fluorescence detected high pressure profiles demonstrate that pressures below 3 kbar result in dissociation of the dimer to a monomeric species that presents no hydrophobic binding sites for ANS. The FTIR-detected high pressure profile obtained under significantly different solution conditions (30 mM trp repressor in absence of denaturant) exhibits a much smaller pressure dependence than the fluorescence detected profiles. The pressure-denatured form obtained under the FTIR conditions retains about 50 % alpha-helical structure. From this we conclude that the secondary structure present in the high pressure state achieved under the conditions of the fluorescence experiments is at least as disrupted as that achieved under FTIR conditions. Fluorescence-detected pressure-jump relaxation studies in the presence of non-denaturing concentrations of GuHCl reveal a positive activation volume for the association/folding reaction and a negative activation volume for dissociation/unfolding reaction, implicating dehydration as the rate-limiting step for association/folding and hydration as the rate-limiting step for unfolding. The GuHCl concentration dependence of the kinetic parameters place the transition state at least half-way along the reaction coordinate between the unfolded and folded states. The temperature dependence of the pressure-jump fluorescence-detected dissociation/unfolding reaction in the presence of non-denaturing GuHCl suggests that the curvature in the temperature dependence of the stability arises from non-Arrhenius behavior of the folding rate constant, consistent with a large decrease in heat capacity upon formation of the transition state from the unfolded state. The decrease in the equilibrium volume change for folding with increasing temperature (due to differences in thermal expansivity of the folded and unfolded states) arises from a decrease in the absolute value for the activation volume for unfolding, thus indicating that the thermal expansivity of the transition state is similar to that of the unfolded state.     

Evidence for Initial Non-specific Polypeptide Chain Collapse During the Refolding of the SH3 Domain of PI3 Kinase

Refolding of the SH3 domain of PI3 kinase from the guanidine hydrochloride (GdnHCl)-unfolded state has been probed with millisecond (stopped flow) and sub-millisecond (continuous flow) measurements of the change in fluorescence, circular dichroism, ANS fluorescence and three-site fluorescence resonance energy transfer (FRET) efficiency. Fluorescence measurements are unable to detect structural changes preceding the rate-limiting step of folding, whereas measurements of changes in ANS fluorescence and FRET efficiency indicate that polypeptide chain collapse precedes the major structural transition. The initial chain collapse reaction is complete within 150 μs. The collapsed form at this time possesses hydrophobic clusters to which ANS binds. Each of the three measured intra-molecular distances has contracted to an extent predicted by the dependence of the FRET signal in completely unfolded protein on denaturant concentration, indicating that contraction is non-specific. The extent of contraction of each intra-molecular distance in the collapsed product of sub-millisecond folding increases continuously with a decrease in [GdnHCl]. The gradual contraction is continuous with the gradual contraction seen in completely unfolded protein, and its dependence on [GdnHCl] is not indicative of an all-or-none collapse reaction. The dependence of the extent of contraction on [GdnHCl] was similar for the three distances, indicating that chain collapse occurs in a synchronous manner across different segments of the polypeptide chain. The sub-millisecond measurements of folding in GdnHCl were unable to determine whether hydrophobic cluster formation, probed by ANS fluorescence measurement, precedes chain contraction probed by FRET. To determine whether hydrogen bonding plays a role in initial chain collapse, folding was initiated by dilution of the urea-unfolded state. The extent of contraction of at least one intra-molecular distance in the collapsed product of sub-millisecond folding in urea is similar to that seen in GdnHCl, and the initial contraction in urea too appears to be gradual.     

A shorter peptide model from staphylococcal nuclease for the folding-unfolding equilibrium of a beta-hairpin shows that unfolded state has significant contribution from compact conformational states

It is important to understand the conformational features of the unfolded state in equilibrium with folded state under physiological conditions. In this paper, we consider a short peptide model LMYKGQPM from staphylococcal nuclease to model the conformational equilibrium between a hairpin conformation and its unfolded state using molecular dynamics simulation under NVT conditions at 300K using GROMOS96 force field. The free energy landscape has overall funnel-like shape with hairpin conformations sampling the minima. The "unfolded" state has a higher free energy of approximately 12kJ/mol with respect to native hairpin minimum and occupies a plateau region. We find that the unfolded state has significant contributions from compact conformations. Many of these conformations have hairpin-like topology. Further, these compact conformational forms are stabilized by hydrophobic interactions. Conversion between native and non-native hairpins occurs via unfolded states. Frequent conversions between folded and unfolded hairpins are observed with single exponential kinetics. We compare our results with the emerging picture of unfolded state from both experimental and theoretical studies.     

NOE data demonstrating a compact unfolded state for an SH3 domain under non-denaturing conditions.

The N-terminal SH3 domain of drk (drkN SH3) is unstable, existing in equilibrium between a folded state (Fexch) and an unfolded state (Uexch) under non-denaturing buffer conditions. Using a15N/2H-labeled sample, long range amide NOEs can be observed in the Uexchstate as a result of reduced relaxation, in some cases correlating protons over 40 residues apart. These long range NOEs disappear upon addition of 2 M guanidinium chloride, demonstrating that there are substantial differences between the Uexchand the guanidine denatured states. Calculations using the long range NOEs of the Uexchstate yield highly compact structures having non-native turns and a non-native buried tryptophan residue. These structures agree with experimental stopped-flow fluorescence data and analytical ultracentrifugation results. Since protein stability depends on the structural and dynamic properties of both the folded and unfolded states, this study provides insights into the stability of the drkN SH3 domain. These results provide the first strong NOE-based evidence for compact unfolded states of proteins and suggest that some unfolded states under physiological conditions have specific interactions leading to compact structures.     

Urea Impedes the Hydrophobic Collapse of Partially Unfolded Proteins

Proteins are denatured in aqueous urea solution. The nature of the molecular driving forces has received substantial attention in the past, whereas the question how urea acts at different phases of unfolding is not yet well understood at the atomic level. In particular, it is unclear whether urea actively attacks folded proteins or instead stabilizes unfolded conformations. Here we investigated the effect of urea at different phases of unfolding by molecular dynamics simulations, and the behavior of partially unfolded states in both aqueous urea solution and in pure water was compared. Whereas the partially unfolded protein in water exhibited hydrophobic collapses as primary refolding events, it remained stable or even underwent further unfolding steps in aqueous urea solution. Further, initial unfolding steps of the folded protein were found not to be triggered by urea, but instead, stabilized. The underlying mechanism of this stabilization is a favorable interaction of urea with transiently exposed, less-polar residues and the protein backbone, thereby impeding back-reactions. Taken together, these results suggest that, quite generally, urea-induced protein unfolding proceeds primarily not by active attack. Rather, thermal fluctuations toward the unfolded state are stabilized and the hydrophobic collapse of partially unfolded proteins toward the native state is impeded. As a result, the equilibrium is shifted toward the unfolded state.     

Insights into conformation and dynamics of protein GB1 during folding and unfolding by NMR

Understanding protein stability requires characterization of structural determinants of the folded and unfolded states. Many proteins are capable of populating partially folded states under specific solution conditions. Occasionally, coexistence of the folded and an unfolded state under non- or mildly denaturing conditions can be observed by NMR, allowing us to structurally probe these states under identical conditions. Here we report on a destabilized mutant of the B1 domain of protein G (GB1) whose equilibrium unfolding was systematically investigated. Backbone amide residual dipolar couplings (RDCs), the tryptophan Nepsilon-H resonance and the amide nitrogen transverse relaxation rates (R2s) for varying pH values and different temperatures were measured. The backbone amide RDCs indicate that prior to complete unfolding, two melting hot spots are formed at the turn around T11, L12 and K13 and the N terminus of the helix at A24 and T25. The RDCs for the low pH, thermally unfolded state of GB1 are very small and do not indicate the presence of any native-like structure. Amide nitrogen transverse relaxation rates for GB1 in the folded state at different temperatures exhibit large contributions from exchange processes and the associated dynamics display considerable heterogeneity. Our data provide clear evidence for intermediate conformations and multi-state equilibrium un/folding for this GB1 variant.     

Kinetics of the Triplex-Duplex Transition in DNA

The kinetics of triplex folding/unfolding is investigated by the single-molecule fluorescence resonance energy transfer (FRET) technique. In neutral pH conditions, the average dwell times in both high-FRET (folded) and low-FRET (unfolded) states are comparable, meaning that the triplex is marginally stable. The dwell-time distributions are qualitatively different: while the dwell-time distribution of the high-FRET state should be fit with at least a double-exponential function, the dwell-time distribution of the low-FRET state can be fit with a single-exponential function. We propose a model where the folding can be trapped in metastable states, which is consistent with the FRET data. Our model also accounts for the fact that the relevant timescales of triplex folding/unfolding are macroscopic.     

Compaction of ribosomal protein S6 by sucrose occurs only under native conditions

The effect of osmolyte sucrose on the stability and compaction of the folded and unfolded states of ribosomal protein S6 from Thermus thermophilus was analyzed. Confirming previous results obtained with sodium sulfate and trehalose, refolding stopped-flow measurements of S6 show that sucrose favors the conversion of the unfolded state ensemble to a highly compact structure (75% as compact as the folded state). This conversion occurs when the unfolded state is suddenly placed under native conditions and the compact state accumulates in a transient off-folding pathway. This effect of sucrose on the compaction of the unfolded state ensemble is counteracted by guanidinium hydrochloride. The compact state does not accumulate at higher guanidinium concentrations and the unfolded state ensemble does not display increased compaction in the presence of 6 M guanidinium as evaluated by collisional quenching of tryptophan fluorescence. In contrast, accessibility of the tryptophan residue of folded S6 above 1 M sucrose concentration decreased as a result of an increased compaction of the folded state. Unfolding stopped-flow measurements of S6 reflect this increased compaction of the folded state, but the unfolding pathway is not affected by sucrose. Compaction of folded and unfolded S6 induced by sucrose occurs under native conditions indicating that decreased protein conformational entropy significantly contributes to the mechanism of protein stabilization by osmolytes.