Origin of unusual phi-values in protein folding: evidence against specific nucleation sites

phi(f)-value analysis is one of the most common methods to characterize the structure of protein folding transition states. It compares the effects of mutations on the folding kinetics with the respective effects on equilibrium stability. The interpretation of the results usually focuses on a few unusual phi(f)-values, which are either particularly high or which are larger than 1 or smaller than 0. These mutations are believed to affect the most important regions for the folding process. A major uncertainty in experimental phi(f)-values is introduced by the commonly used analysis of only a single mutant at various positions in a protein (two-point analysis). To test the reliability of two-point phi(f)-values we used reference data from three positions in two different proteins at which multiple mutations have been introduced. The results show that two-point phi(f)-values are highly inaccurate if the difference in stability between two variants is less than 7 kJ/mol, corresponding to a 20-fold difference in equilibrium constant. Comparison with reported phi(f)-values for 11 proteins shows that most unusual phi(f)-values are observed in mutants which show changes in protein stability that are too small to allow a reliable analysis. The results argue against specific nucleation sites in protein folding and give a picture of transition states as distorted native states for the major part of a protein or for large substructures.     

Evidence for sequential barriers and obligatory intermediates in apparent two-state protein folding

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.     

Burst-phase expansion of native protein prior to global unfolding in SDS

Although numerous studies have been directed at understanding early folding events through the characterization of folding intermediates, there are few reports on the very late folding events, i.e. on the events taking place on the native side of the folding barrier and on alternative conformations of the folded state. To shed further light on these issues, we have characterized by protein engineering the structure of an expanded but native-like intermediate that accumulates transiently in the unfolding reaction of the small protein S6 in the presence of SDS. The results show that the SDS micelles attack the native protein in the dead-time of the denaturation experiment, causing an expansion of the hydrophobic core prior to the major unfolding transition. We distinguish two forms of the unfolding intermediate that are correlated with the micellar structure. With spherical micelles, the expansion is seen mainly as a weakening of the interactions which anchor the two alpha-helices to the core of the S6 structure. With cylindrical micelles, prevalent at higher SDS concentrations, the expansion is more global and produces a species which closely resembles the transition-state structure for unfolding in GdmCl. Despite the highly weakened core, the micelle-associated intermediate displays cooperative unfolding, indicating a significant structural plasticity of the species on the native side of the folding barrier in the presence of SDS.     

On the role of some conserved and nonconserved amino acid residues in the transitional state and intermediate of apomyoglobin folding

The contributions of some amino acid residues in the A, B, G, and H helices to the formation of the folding nucleus and folding intermediate of apomyoglobin were estimated. The effects of point substitutions of Ala for hydrophobic amino acid residues on the structural stability of the native (N) protein and its folding intermediate (I), as well as on the folding/unfolding rates for four mutant apomyoglobin forms, were studied. The equilibrium and kinetic studies of the folding/unfolding rates of these mutant proteins in a wide range of urea concentrations demonstrated that their native state was considerably destabilized as compared with the wild-type protein, whereas the stability of the intermediate state changed moderately. It was shown that the amino acid residues in the A, G, and H helices contributed insignificantly to the stabilization of the apomyoglobin folding nucleus in the rate-limiting I ? N transition, taking place after the formation of the intermediate, whereas the residue of the B helix was of great importance in the formation of the folding nucleus in this transition.     

Comparison of the transition states for folding of two Ig-like proteins from different superfamilies

In the "fold approach" proteins with a similar fold but different sequences are compared in order to investigate the relationship between native state structure and folding behaviour. Here we compare the properties of the transition states for folding of TI I27, the 27th immunoglobulin domain from human cardiac titin, and that of TNfn3, the third fibronectin type III domain from human tenascin. Experimental phi-values were used as restraints in molecular dynamics simulations to determine the structures that make up the transition state ensembles (TSEs) for folding of the two proteins. The restrained simulations that we present allow a detailed structural comparison of the two TSEs to be made. Further calculations show explicitly that for both proteins the formation of the interactions involving the residues in the folding nucleus is sufficient for the establishment of the topology of the Ig-like fold. We found that, although the folding nuclei of the two proteins are similar, the packing of the folding nucleus of TI I27 is much tighter than that of TNfn3, reflecting the higher experimental phi-values and beta(T) (Tanford Beta) of TI I27. These results suggest that the folding nucleus can be significantly deformed to accommodate extensive sequence variation while conserving the same folding mechanism.     

The transition state for folding of a peripheral subunit-binding domain contains robust and ionic-strength dependent characteristics

The denaturant dependencies of the folding and unfolding kinetics were used to characterize the structure of the transition state for folding of E3BD, a peripheral subunit-binding domain. For the majority of E3BD mutants, the Phi-values calculated at 298 K from the analysis of chevron plots were in good agreement with those previously determined at 325 K using Arrhenius analysis. This agreement further demonstrates the general robustness of Phi-value analyses, since different experiments, methods of denaturation and thermodynamic assumptions were used to determine each set of Phi(F) values. The structure of the transition state for folding was grossly conserved at 298 K and 325 K, with residues in Helix I playing a lesser role in folding than those located in the 3(10) helix, disordered loop and Helix II. However, the energetic contributions of a cluster of basic residues close to the N-terminus and Helix I, which are an integral part of the ligand-binding site, were susceptible to ionic strength effects because of electrostatic strain in native and transition states of E3BD at low ionic strength. We found no evidence of the downhill folding previously proposed for E3BD, even though the conditions employed in this study significantly increased the energetic bias towards the native state.     

Simulation and experiment conspire to reveal cryptic intermediates and a slide from the nucleation-condensation to framework mechanism of folding

There is a change from three-state to two-state kinetics of folding across the homeodomain superfamily of proteins as the mechanism slides from framework to nucleation-condensation. The tendency for framework folding in this family correlates with inherent helical propensity. The cellular myeloblastis protein (c-Myb) falls in the mechanistic transition region. An earlier, preliminary report of protein engineering experiments and molecular dynamics simulations (MD) showed that the folding mechanism for this protein has aspects of both the nucleation-condensation and framework models. In the more in-depth analysis of the MD trajectories presented here, we find that folding may be attributed to both of these mechanisms in different regions of the protein. The folding of the loop, middle helix, and turn is best described by nucleation-condensation, whereas folding of the N and C-terminal helices may be described by the framework model. Experimentally, c-Myb folds by apparent two-state kinetics, but the MD simulations predict that the kinetics hide a high-energy intermediate. We stabilized this hypothetical folding intermediate by deleting a residue (P174) in the loop between its second and third helices, and the mutant intermediate is long-lived in the simulations. Equilibrium and kinetic experiments demonstrate that folding of the DeltaP174 mutant is indeed three-state. The presence and shape of the intermediate observed in the simulations were confirmed by small angle X-ray scattering experiments.     

Hammond behavior versus ground state effects in protein folding: evidence for narrow free energy barriers and residual structure in unfolded states

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.     

Structure of stable protein folding intermediates by equilibrium phi-analysis: the apoflavodoxin thermal intermediate

Protein intermediates in equilibrium with native states may play important roles in protein dynamics but, in cases, can initiate harmful aggregation events. Investigating equilibrium protein intermediates is thus important for understanding protein behaviour (useful or pernicious) but it is hampered by difficulties in gathering structural information. We show here that the phi-analysis techniques developed to investigate transition states of protein folding can be extended to determine low-resolution three-dimensional structures of protein equilibrium intermediates. The analysis proposed is based solely on equilibrium data and is illustrated by determination of the structure of the apoflavodoxin thermal unfolding intermediate. In this conformation, a large part of the protein remains close to natively folded, but a 40 residue region is clearly unfolded. This structure is fully consistent with the NMR data gathered on an apoflavodoxin mutant designed specifically to stabilise the intermediate. The structure shows that the folded region of the intermediate is much larger than the proton slow-exchange core at 25 degrees C. It also reveals that the unfolded region is made of elements whose packing surface is more polar than average. In addition, it constitutes a useful guide to rationally stabilise the native state relative to the intermediate state, a far from trivial task.     

Local interactions and the optimization of protein folding

The role of local interactions in protein folding has recently been the subject of some controversy. Here we investigate an extension of Zwanzig's simple and general model of folding in which local and nonlocal interactions are represented by functions of single and multiple conformational degrees of freedom, respectively. The kinetics and thermodynamics of folding are studied for a series of energy functions in which the energy of the native structure is fixed, but the relative contributions of local and nonlocal interactions to this energy are varied over a broad range. For funnel shaped energy landscapes, we find that 1) the rate of folding increases, but the stability of the folded state decreases, as the contribution of local interactions to the energy of the native structure increases, and 2) the amount of native structure in the unfolded state and the transition state vary considerably with the local interaction strength. Simple exponential kinetics and a well-defined free energy barrier separating folded and unfolded states are observed when nonlocal interactions make an appreciable contribution to the energy of the native structure; in such cases a transition state theory type approximation yields reasonably accurate estimates of the folding rate. Bumps in the folding funnel near the native state, which could result from desolvation effects, side chain freezing, or the breaking of nonnative contacts, significantly alter the dependence of the folding rate on the local interaction strength: the rate of folding decreases when the local interaction strength is increased beyond a certain point. A survey of the distribution of strong contacts in the protein structure database suggests that evolutionary optimization has involved both kinetics and thermodynamics: strong contacts are enriched at both very short and very long sequence separations. Proteins 29:282–291, 1997. © 1997 Wiley-Liss, Inc.     

The structure of the major transition state for folding of an FF domain from experiment and simulation

We have analysed the transition state of folding of the four-helix FF domain from HYPA/FBP11 by high-resolution experiment and simulation as part of a continuing effort to understand the principles of folding and the refinement of predictive methods. The major transition state for folding was subjected to a Phi-value analysis utilising 50 mutants. The transition state contained a nucleus for folding centred around the end of helix 1 (H1) and the beginning of helix 2 (H2). Secondary structure in this region was fully formed (PhiF=0.9-1) and tertiary interactions were well developed. Interactions in the distal part of the native structure were weak (PhiF=0-0.2). The hydrophobic core and other parts of the protein displayed intermediate Phi-values, suggesting that interactions coalesce as the end of H1 and beginning of H2 are in the process of being formed. The distribution of Phi-values resembled that of barnase, which folds via an intermediate, rather than that of CI2 which folds by a concerted nucleation-condensation mechanism. The overall picture of the transition state structure identified in molecular dynamics simulations is in qualitative agreement, with the turn connecting H1 and H2 being formed, a loosened core, and H4 partially unfolded and detached from the core. There are some differences in the details and interpretation of specific Phi-values.     

Dynamics of hydrogen bond desolvation in protein folding

As proteins fold, a progressive structuring, immobilization and eventual exclusion of water surrounding backbone hydrogen bonds takes place. This process turns hydrogen bonds into major determinants of the folding pathway and compensates for the penalty of desolvation of the backbone polar groups. Taken as an average over all hydrogen bonds in a native fold, this extent of protection is found to be nearly ubiquitous. It is dynamically crucial, determining a constraint in the long-time limit behavior of coarse-grained ab initio simulations. Furthermore, an examination of one of the longest available (1micros) all-atom simulations with explicit solvent reveals that this average extent of protection is a constant of motion for the folding trajectory. We propose how such a stabilization is best achieved by clustering five hydrophobes around the backbone hydrogen bonds, an arrangement that yields the optimal stabilization. Our results support and clarify the view that hydrophobic surface burial should be commensurate with hydrogen-bond formation and enable us to define a basic desolvation motif inherent to structure and folding dynamics.     

The folding transition state of the cold shock protein is strongly polarized

It has been shown recently that an 11-residue peptide fragment of transthyretin, TTR(105-115), can form amyloid fibrils in vitro by adopting an extended beta-strand conformation. We used molecular dynamics simulations on systems of TTR(105-115) peptides, for a total length of about 5 micros, to explore the process of self-assembly and the structures of the resulting aggregates. Our results suggest that an antiparallel association of the beta-strands is more probable than a parallel one and that the central residues (T106-L111) in a beta-strand have a high propensity to form inter-peptide hydrogen bonds. The study of the dynamics of self-association indicated that, for this peptide, trajectories leading to conformations with high alpha-helical content are off-pathway from those leading to aggregates with high beta-structure content. We also show that the diverse oligomeric structures that form spontaneously in the molecular dynamics simulations are, to a large extent, compatible with solid-state NMR experimental measurements, including chemical shifts, on fully formed fibrils. The strategy that we present may therefore be used in the design of new experiments to determine the structure of amyloid fibrils, such as those involving site-specific isotope labelling schemes to measure key inter-atomic distances.     

Consistency in structural energetics of protein folding and peptide recognition.

We report a new free energy decomposition that includes structure-derived atomic contact energies for the desolvation component, and show that it applies equally well to the analysis of single-domain protein folding and to the binding of flexible peptides to proteins. Specifically, we selected the 17 single-domain proteins for which the three-dimensional structures and thermodynamic unfolding free energies are available. By calculating all terms except the backbone conformational entropy change and comparing the result to the experimentally measured free energy, we estimated that the mean entropy gain by the backbone chain upon unfolding (delta Sbb) is 5.3 cal/K per mole of residue, and that the average backbone entropy for glycine is 6.7 cal/K. Both numbers are in close agreement with recent estimates made by entirely different methods, suggesting a promising degree of consistency between data obtained from disparate sources. In addition, a quantitative analysis of the folding free energy indicates that the unfavorable backbone entropy for each of the proteins is balanced predominantly by favorable backbone interactions. Finally, because the binding of flexible peptides to receptors is physically similar to folding, the free energy function should, in principle, be equally applicable to flexible docking. By combining atomic contact energies, electrostatics, and sequence-dependent backbone entropy, we calculated a priori the free energy changes associated with the binding of four different peptides to HLA-A2, 1 MHC molecule and found agreement with experiment to within 10% without parameter adjustment.     

Kinetic evidence of an on-pathway intermediate in the folding of lysozyme

By means of a kinetic test, it was demonstrated that one of the folding intermediates (Ialpha) of hen lysozyme with alpha-domain folded and beta-domain unfolded is on the folding pathway under the classical definition. Ialpha folds to the native (N) state directly (unfolded (U) <==> Ialpha <==> N) without having to unfold to U and then refold to N through alternative folding pathways as in Ialpha <==> U <==> N.     

Effects of the difference in the unfolded-state ensemble on the folding of Escherichia coli dihydrofolate reductase

The unfolded state of a protein is an ensemble of a large number of conformations ranging from fully extended to compact structures. To investigate the effects of the difference in the unfolded-state ensemble on protein folding, we have studied the structure, stability, and folding of "circular" dihydrofolate reductase (DHFR) from Escherichia coli in which the N and C-terminal regions are cross-linked by a disulfide bond, and compared the results with those of disulfide-reduced "linear" DHFR. Equilibrium studies by circular dichroism, difference absorption spectra, solution X-ray scattering, and size-exclusion chromatography show that whereas the native structures of both proteins are essentially the same, the unfolded state of circular DHFR adopts more compact conformations than the unfolded state of the linear form, even with the absence of secondary structure. Circular DHFR is more stable than linear DHFR, which may be due to the decrease in the conformational entropy of the unfolded state as a result of circularization. Kinetic refolding measurements by stopped-flow circular dichroism and fluorescence show that under the native conditions both proteins accumulate a burst-phase intermediate having the same structures and both fold by the same complex folding mechanism with the same folding rates. Thus, the effects of the difference in the unfolded state of circular and linear DHFRs on the refolding reaction are not observed after the formation of the intermediate. This suggests that for the proteins with close termini in the native structure, early compaction of a protein molecule to form a specific folding intermediate with the N and C-terminal regions in close proximity is a crucial event in folding. If there is an enhancement in the folding reflecting the reduction in the breadth of the unfolded-state ensemble for circular DHFR, this acceleration must occur in the sub-millisecond time-range.     

Fine structure analysis of a protein folding transition state; distinguishing between hydrophobic stabilization and specific packing

Developing a detailed understanding of the structure and energetics of protein folding transition states is a key step in describing the folding process. The phi-value analysis approach allows the energetic contribution of side-chains to be mapped out by comparing wild-type with individual mutants where conservative changes are introduced. Studies where multiple substitutions are made at individual sites are much rarer but are potentially very useful for understanding the contribution of each element of a side-chain to transition state formation, and for distinguishing the relative importance of specific packing versus hydrophobic interactions. We have made a series of conservative mutations at multiple buried sites in the N-terminal domain of L9 in order to assess the relative importance of specific side-chain packing versus less specific hydrophobic stabilization of the transition state. A total of 28 variants were prepared using both naturally occurring and non-naturally occurring amino acids at six sites. Analysis of the mutants by NMR and CD showed no perturbation of the structure. There is no correlation between changes in hydrophobicity and changes in stability. In contrast, there is excellent linear correlation between the hydrophobicity of a side-chain and the log of the folding rate, ln(k(f)). The correlation between ln(k(f)) and the change in hydrophobicity holds even for substitutions that change the shape and/or size of a side-chain significantly. For most sites, the correlation with the logarithm of the unfolding rate, ln(k(u)), is much worse. Mutants with more hydrophobic amino acid substitutions fold faster, and those with less hydrophobic amino acid substitutions fold slower. The results show that hydrophobic interactions amongst core residues are an important driving force for forming the transition state, and are more important than specific tight packing interactions. Finally, a number of substitutions lead to negative phi-values and the origin of these effects are described.