Non-linear rate-equilibrium free energy relationships and Hammond behavior in protein folding

Non-linear rate-equilibrium relationships upon mutation or changes in solvent conditions are frequently observed in protein folding reactions and are usually interpreted in terms of Hammond behavior. Here we first give a general overview over the concept of transition state movements in chemical reactions and discuss its application to protein folding. We then show examples for genuine Hammond behavior and for apparent transition state movements caused by other effects like changes in the rate-limiting step of the folding reaction or ground state effects, i.e. structural changes in either the native state or the unfolded state. These examples show that apparent transition state movements can easily be mistaken for Hammond behavior. We describe experimental tests using self- and cross-interaction parameters to distinguish between structural changes in a single transition state following Hammond behavior and apparent transition state movements caused by other effects.     

Shape of the free energy barriers for protein folding probed by multiple perturbation analysis

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.     

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.     

Transient structure formation in unfolded acyl-coenzyme A-binding protein observed by site-directed spin labelling

Paramagnetic relaxation has been used to monitor the formation of structure in the folding peptide chain of guanidinium chloride-denatured acyl-coenzyme A-binding protein. The spin label (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate (MTSL) was covalently bound to a single cysteine residue introduced into five different positions in the amino acid sequence. It was shown that the formation of structure in the folding peptide chain at conditions where 95% of the sample is unfolded brings the relaxation probe close to a wide range of residues in the peptide chain, which are not affected in the native folded structure. It is suggested that the experiment is recording the formation of many discrete and transient structures in the polypeptide chain in the preface of protein folding. Analysis of secondary chemical shifts shows a high propensity for alpha-helix formation in the C-terminal part of the polypeptide chain, which forms an alpha-helix in the native structure and a high propensity for turn formation in two regions of the polypeptide that form turns in the native structure. The results contribute to the idea that native-like structural elements form transiently in the unfolded state, and that these may be of importance to the initiation of protein folding.     

Conformational plasticity in folding of the split beta-alpha-beta protein S6: evidence for burst-phase disruption of the native state

An increasing number of folding studies of two-state proteins shows that point mutations sometimes change the kinetic m-values, leading to kinks and curves in the chevron plots. The molecular origin of these changes is yet unclear although it is speculated that they are linked to structural rearrangement of the transition state or to accumulation of meta-stable intermediates. To shed more light on this issue, we present here a combined m and phi-value analysis of the split beta-alpha-beta protein S6. Wild-type S6 displays classical two-state kinetics with v-shaped chevron plot, but a majority of its mutants display distinct m-value changes or curved chevrons. We observe that this kinetic aberration of S6 is linked to mutations that are clustered in distinct regions of the native structure. The most pronounced changes, i.e. decrease in the m-value for the unfolding rate constant, are seen upon truncation of interactions between the N and C termini, whereas mutations in the centre of the hydrophobic core show smaller or even opposed effects. As a consequence, the calculated phi-values display a systematic increase upon addition of denaturant. In the case of S6, the phenomenon seems to arise from a general plasticity of the different species on the folding pathway. That is, the structure of the denatured ensemble, the transition state, and the native ground-state for unfolding seem to change upon mutation. From these changes, it is concluded that interactions spanning the centre of the hydrophobic core form early in folding, whereas the entropically disfavoured interactions linking the N and C termini consolidate very late, mainly on the down-hill-side of the folding barrier.     

Dynamics of unfolded polypeptide chains as model for the earliest steps in protein folding

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 5 ns for flexible glycine-rich chains and of 12 ns 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.     

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.     

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.     

Role of unfolded state heterogeneity and en-route ruggedness in protein folding kinetics

In order to improve our understanding of the physical bases of protein folding, there is a compelling need for better connections between experimental and computational approaches. This work addresses the role of unfolded state conformational heterogeneity and en-route intermediates, as an aid for planning and interpreting protein folding experiments. The expected kinetics were modeled for different types of energy landscapes, including multiple parallel folding routes, preferential paths dominated by one primary folding route, and distributed paths with a wide spectrum of microscopic folding rate constants. In the presence of one or more preferential routes, conformational exchange among unfolded state populations slows down the observed rates for native protein formation. We find this to be a general phenomenon, taking place even when unfolded conformations interconvert much faster than the "escape" rate constants to folding. Dramatic kinetic deceleration is expected in the presence of an increasing number of folding-incompetent unfolded conformations. This argues for the existence of parallel folding paths involving several folding-competent unfolded conformations, during the early stages of protein folding. Deviations from single-exponential behavior are observed for unfolded conformations exchanging at comparable rates or more slowly than folding events. Analysis of the effect of en-route (on-path) intermediate formation and landscape ruggedness on folding kinetics leads to the following unexpected conclusions: (1) intermediates, which often retard native state formation, may in some cases accelerate folding, and (2) rugged landscapes, usually associated with stretched exponentials, display single-exponential behavior in the presence of late high-friction paths.     

Polyproline II helical structure in protein unfolded states: lysine peptides revisited

The left-handed polyproline II (PPII) helix gives rise to a circular dichroism spectrum that is remarkably similar to that of unfolded proteins. This similarity has been used as the basis for the hypothesis that unfolded proteins possess considerable PPII helical content. It has long been known that homopolymers of lysine adopt the PPII helical conformation at neutral pH, presumably a result of electrostatic repulsion between side chains. It is shown here that a seven-residue lysine peptide also adopts the PPII conformation. In contrast with homopolymers of lysine, this short peptide is shown to retain PPII helical character under conditions in which side-chain charges are heavily screened or even neutralized. The most plausible explanation for these observations is that the peptide backbone favors the PPII conformation to maximize favorable interactions with solvent. These data are evidence that unfolded proteins do indeed possess PPII content, indicating that the ensemble of unfolded states is significantly smaller than is commonly assumed.     

Direct characterization of the folded, unfolded and urea-denatured states of the C-terminal domain of the ribosomal protein L9

The stability of the isolated C-terminal domain of the ribosomal protein L9 (CTL9) is strongly dependent upon pH. Below pH 4.2, the folded and unfolded states are both populated significantly. Their interconversion is slow on the NMR chemical shift time-scale and separate, well-resolved resonances from each state are observed. This allows the hydrodynamic properties of both states to be studied under identical conditions by using pulse field gradient NMR experiments. Hydrodynamic radii of the folded, unfolded and urea denatured protein molecules at pD 3.8 have been derived. The acid-denatured protein has a significantly smaller hydrodynamic radius, 28.2A, compared to that of the urea-denatured protein, which is 33.6A at pD 3.8. Far-UV CD spectra show that there is more residual secondary structure retained in the acid-denatured ensemble than in the urea-denatured one. ANS binding experiments and analysis of the CD data show that this acid-denatured species is not a molten globule state. Diffusion measurements of CTL9 were conducted over the pD range from 2.1 to 7.0. The hydrodynamic radii of both the folded and the acid-unfolded protein start to increase below pD 4, with the radius of hydration of the acid-unfolded state increasing from 25.1A at pD 4.2 to 33.5A at pD 2.1. The hydrodynamic radius of the urea-denatured protein is much less sensitive to pH. The unfolded protein at pD 2.1, no urea, has almost the same hydrodynamic radius as the urea-denatured protein at pD 3.8. The CD spectra, however, show significant differences in residual secondary structure, and the acid-denatured state contains more structure.     

Native state energetics of the Src SH2 domain: evidence for a partially structured state in the denatured ensemble

We have defined the free-energy profile of the Src SH2 domain using a variety of biophysical techniques. Equilibrium and kinetic experiments monitored by tryptophan fluorescence show that Src SH2 is quite stable and folds rapidly by a two-state mechanism, without populating any intermediates. Native state hydrogen-deuterium exchange confirms this two-state behavior; we detect no cooperative partially unfolded forms in equilibrium with the native conformation under any conditions. Interestingly, the apparent stability of the protein from hydrogen exchange is 2 kcal/mol greater than the stability determined by both equilibrium and kinetic studies followed by fluorescence. Native-state proteolysis demonstrates that this "super protection" does not result from a deviation from the linear extrapolation model used to fit the fluorescence data. Instead, it likely arises from a notable compaction in the unfolded state under native conditions, resulting in an ensemble of conformations with substantial solvent exposure of side chains and flexible regions sensitive to proteolysis, but backbone amides that exchange with solvent approximately 30-fold slower than would be expected for a random coil. The apparently simple behavior of Src SH2 in traditional unfolding studies masks the significant complexity present in the denatured-state ensemble.     

Denatured collapsed states in protein folding: example of apomyoglobin.

Experimental approaches, including circular dichroism, small angle X-ray scattering, steady-state fluorescence, and fluorescence energy transfer, were applied to study the 3D-structure of apomyolgobin in different conformational states. These included the native and molten globules, along with either less ordered conformations induced by the addition of anions or completely unfolded states. The results show that the partially folded forms of apomyoglobin stabilized by KCl and/or Na(2)SO(4) under unfolding conditions (pH 2) exhibit a significant amount of secondary structure (circular dichroism), low packing density of protein molecules (SAXS), and native-like dimensions of the AGH core (fluorescence energy transfer). This finding indicates that a native-like tertiary fold of the polypeptide chain, i.e., the spatial organization of secondary structure elements, most likely emerges prior to the formation of the molten globule state.     

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.     

Structural analysis of the rate-limiting transition states in the folding of Im7 and Im9: similarities and differences in the folding of homologous proteins

The bacterial immunity proteins Im7 and Im9 fold with mechanisms of different kinetic complexity. Whilst Im9 folds in a two-state transition at pH 7.0 and 10 degrees C, Im7 populates an on-pathway intermediate under these conditions. In order to assess the role of sequence versus topology in the folding of these proteins, and to analyse the effect of populating an intermediate on the landscape for folding, we have determined the conformational properties of the rate-limiting transition state for Im9 folding/unfolding using Phi(F)-value analysis and have compared the results with similar data obtained previously for Im7. The data show that the rate-limiting transition states for Im9 and Im7 folding/unfolding are similar: both are compact (beta(T)=0.94 and 0.89, respectively) and contain three of the four native helices docked around a specific hydrophobic core. Significant differences are observed, however, in the magnitude of the Phi(F)-values obtained for the two proteins. Of the 20 residues studied in both proteins, ten have Phi(F)-values in Im7 that exceed those in Im9 by more than 0.2, and of these five differ by more than 0.4. The data suggest that the population of an intermediate in Im7 results in folding via a transition state ensemble that is conformationally restricted relative to that of Im9. The data are consistent with the view that topology is an important determinant of folding. Importantly, however, they also demonstrate that while the folding transition state may be conserved in homologous proteins that fold with two and three-state kinetics, the population of an intermediate can have a significant effect on the breadth of the transition state ensemble.