Thermodynamics and kinetics of non-native interactions in protein folding: a single point mutant significantly stabilizes the N-terminal domain of L9 by modulating non-native interactions in the denatured state

Comparatively little is known about the role of non-native interactions in protein folding and their role in both folding and stability is controversial. We demonstrate that non-native electrostatic interactions involving specific residues in the denatured state can have a significant effect upon protein stability and can persist in the transition state for folding. Mutation of a single surface exposed residue, Lys12 to Met, in the N-terminal domain of the ribosomal protein L9 (NTL9), significantly increased the stability of the protein and led to faster folding. Structural and energetic studies of the wild-type and K12M mutant show that the 1.9 kcal mol(-1) increase in stability is not due to native state effects, but rather is caused by modulation of specific non-native electrostatic interactions in the denatured state. pH dependent stability measurements confirm that the increased stability of the K12M is due to the elimination of favorable non-native interactions in the denatured state. Kinetic studies show that the non-native electrostatic interactions involving K12 persist in the transition state. The analysis demonstrates that canonical Phi-values can arise from the disruption of non-native interactions as well as from the development of native interactions.     

Influence of denatured and intermediate states of folding on protein aggregation

We simulate the aggregation thermodynamics and kinetics of proteins L and G, each of which self-assembles to the same alpha/beta [corrected] topology through distinct folding mechanisms. We find that the aggregation kinetics of both proteins at an experimentally relevant concentration exhibit both fast and slow aggregation pathways, although a greater proportion of protein G aggregation events are slow relative to those of found for protein L. These kinetic differences are correlated with the amount and distribution of intrachain contacts formed in the denatured state ensemble (DSE), or an intermediate state ensemble (ISE) if it exists, as well as the folding timescales of the two proteins. Protein G aggregates more slowly than protein L due to its rapidly formed folding intermediate, which exhibits native intrachain contacts spread across the protein, suggesting that certain early folding intermediates may be selected for by evolution due to their protective role against unwanted aggregation. Protein L shows only localized native structure in the DSE with timescales of folding that are commensurate with the aggregation timescale, leaving it vulnerable to domain swapping or nonnative interactions with other chains that increase the aggregation rate. Folding experiments that characterize the structural signatures of the DSE, ISE, or the transition state ensemble (TSE) under nonaggregating conditions should be able to predict regions where interchain contacts will be made in the aggregate, and to predict slower aggregation rates for proteins with contacts that are dispersed across the fold. Since proteins L and G can both form amyloid fibrils, this work also provides mechanistic and structural insight into the formation of prefibrillar species.     

Charge-charge interactions in the denatured state influence the folding kinetics of ribonuclease Sa

Gaining a better understanding of the denatured state ensemble of proteins is important for understanding protein stability and the mechanism of protein folding. We studied the folding kinetics of ribonuclease Sa (RNase Sa) and a charge-reversal variant (D17R). The refolding kinetics are similar, but the unfolding rate constant is 10-fold greater for the variant. This suggests that charge-charge interactions in the denatured state and the transition state ensembles are more favorable in the variant than in RNase Sa, and shows that charge-charge interactions can influence the kinetics and mechanism of protein folding.     

Charge-charge interactions influence the denatured state ensemble and contribute to protein stability

Several recent studies have shown that it is possible to increase protein stability by improving electrostatic interactions among charged groups on the surface of the folded protein. However, the stability increases are considerably smaller than predicted by a simple Coulomb's law calculation, and in some cases, a charge reversal on the surface leads to a decrease in stability when an increase was predicted. These results suggest that favorable charge-charge interactions are important in determining the denatured state ensemble, and that the free energy of the denatured state may be decreased more than that of the native state by reversing the charge of a side chain. We suggest that when the hydrophobic and hydrogen bonding interactions that stabilize the folded state are disrupted, the unfolded polypeptide chain rearranges to compact conformations with favorable long-range electrostatic interactions. These charge-charge interactions in the denatured state will reduce the net contribution of electrostatic interactions to protein stability and will help determine the denatured state ensemble. To support this idea, we show that the denatured state ensemble of ribonuclease Sa is considerably more compact at pH 7 where favorable charge-charge interactions are possible than at pH 3, where unfavorable electrostatic repulsion among the positive charges causes an expansion of the denatured state ensemble. Further support is provided by studies of the ionic strength dependence of the stability of charge-reversal mutants of ribonuclease Sa. These results may have important implications for the mechanism of protein folding.     

Engineering stabilising beta-sheet interactions into a conformationally flexible region of the folding transition state of ubiquitin

Protein engineering studies suggest that the transition state for the folding of ubiquitin is highly polarised towards the N-terminal part of the sequence and involves a nucleus of residues within the beta-hairpin (residues 1-17) and main alpha-helix (residues 23-34). In contrast, the observation of small phi-values for residues in the C-terminal portion of the sequence (residues 35-76), coupled with a folding topology that results in a much higher contact order, suggests that fast folding of ubiquitin is dependent upon configurational flexibility in the C-terminal part of the polypeptide chain to ensure passage down a relatively smooth folding funnel to the native state. We show that the introduction of a small mini-hairpin motif as an extension of the native 43-50 hairpin stabilises local interactions in the C-terminal part of the sequence, resulting largely in a deceleration of the unfolding kinetics without perturbing the apparent two-state folding mechanism. However, a single-point Leu-->Phe substitution within the engineered hairpin sequence leads to the premature collapse of the denatured ensemble through the stabilisation of non-native interactions and the population of a compact intermediate. Non-linear effects in the kinetic data at low concentrations of denaturant suggest that the collapsed state, which is further stabilised in the presence of cosmotropic salts, may subsequently fold directly to the native state through a "triangular" reaction scheme involving internal rearrangement rather than unfolding and refolding.     

Kinetic analysis of molecular dynamics simulations reveals changes in the denatured state and switch of folding pathways upon single-point mutation of a beta-sheet miniprotein

The effects of a single-point mutation on folding thermodynamics and kinetics are usually interpreted by focusing on the native structure and the transition state. Here, the entire conformational spaces of a 20-residue three-stranded antiparallel beta-sheet peptide (double hairpin) and of its single-point mutant W10V are sampled close to the melting temperature by equilibrium folding-unfolding molecular dynamics simulations for a total of 40 micros. The folded state as well as the most populated free energy basins in the denatured state are isolated by grouping conformations according to fast relaxation at equilibrium. Such kinetic analysis provides more detailed and useful information than a simple projection of the free energy. The W10V mutant has the same native structure as the wild type peptide, and similar folding rate and stability. In the denatured state, the N-terminal hairpin is about 20% more structured in W10V than the wild type mainly because of van der Waals interactions. Notably, the W10V mutation influences also the van der Waals energy at the transition state ensemble causing a shift in the ratio of fluxes between two different transition state regions on parallel folding pathways corresponding to nucleation at either of the two beta-hairpins. Previous experimental studies have focused on the effects of denaturant-dependent or temperature-dependent changes in the structure of the denatured state. The atomistic simulations show that a single-point mutation in the central strand of a beta-sheet peptide results in remarkable changes in the topography of the denatured state ensemble. These changes modulate the relative accessibility of parallel folding pathways because of kinetic partitioning of the denatured state. Therefore, the observed dependence of the folding process on the starting ensemble raises questions on the biological significance of in vitro folding studies under strongly denaturing conditions.     

Methionine oxidation of monomeric lambda repressor: the denatured state ensemble under nondenaturing conditions

Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric lambda repressor and predominantly populate the denatured state under nondenaturing buffer conditions. The denatured ensemble populated under these conditions comprises conformations that are compact. Analytical ultracentrifugation sedimentation velocity experiments indicate a small increase in Stokes radius over that of the native state. A significant degree of alpha-helical structure in these conformations is detected by far-UV circular dichroism, and some tertiary interactions are suggested by near-UV circular dichroism. The characteristics of the denatured state populated by methionine oxidation in nondenaturing buffer are very different from those found in chemical denaturant.     

Analysis of the pH-dependent folding and stability of histidine point mutants allows characterization of the denatured state and transition state for protein folding

pH-Dependent studies of the folding kinetics and stability of a set of His to Gln point mutants were used to characterize the denatured state and transition state ensembles for the C-terminal domain of the ribosomal protein L9 (CTL9). CTL9 contains three histidine residues, two of which, H106 and H134, are buried in the native state, while the third, H144, is more exposed. Comparison of the pH-dependent stability calculated using the Tanford-Wyman linkage relationship to the measured values demonstrates that the apparent pK(a) values of the three histidine residues are not significantly perturbed in the denatured state ensemble. Kinetic measurements show that mutation of H134 has a larger effect on the folding process than does mutation of H106 and H144. The Phi-value for H134 is significantly larger than the Phi-values for the other histidine residues, which are near zero at both pH 5.45 and pH 8.0. The Phi-value for H134 is higher, 0.55, at pH 8.0 than at pH 5.45, 0.39. At pH 5.45, H134 is protonated in the unfolded state but deprotonated in the native state, while at pH 8.0 it is deprotonated in both. There is an excellent linear relationship between stability (logK) and folding rates (logk(f)) over the range of pH 5-9 for all mutants. From these plots, the ratio of DeltaQ( not equal)/DeltaQ can be calculated for each mutant. DeltaQ( not equal) is the difference in the number of protons bound to the transition state and to the unfolded state, while DeltaQ represents the difference between folded and denatured state. The linear plots indicate that the relative position of the transition state ensemble as judged by DeltaQ( not equal)/DeltaQ is independent of pH. The linkage analysis is consistent with the Phi-value analysis, showing that H134 is the most critical contributor to the development of pH-dependent interactions, including desolvation effects in the transition state ensemble.     

Stabilization of the ribosomal protein S6 by trehalose is counterbalanced by the formation of a putative off-pathway species

The effect of trehalose on folding and stability of the small ribosomal protein S6 was studied. Non-disruptive point mutations distributed along the protein structure were analyzed to characterize the stabilizing effect of trehalose and map the folding pathway of S6. On average, the stability of the wild-type and S6 mutants increases by 3 kcal/mol M trehalose. Despite the non-specific thermodynamic stabilization mechanism, trehalose particularly stabilizes the less destabilized mutants. Folding/unfolding kinetics shows clearly that trehalose induces the collapse of the unfolded state to an off-pathway intermediate with non-native diffuse contacts. This state is similar to the collapsed state induced by high concentrations of stabilizing salts, as previously reported. Although it leads to the accumulation of this off-pathway intermediate, trehalose does not change the compactness of the transition state ensemble. Furthermore, the productive folding pathway of S6 is not affected by trehalose as shown by a Phi-value analysis. The unfolded state ensemble of S6 should be more compact in the presence of trehalose and therefore destabilized due to decreased conformational entropy. Increased compaction of the unfolded state ensemble might also occur for more stable mutants of S6, thus explaining the synergistic effect of trehalose and point mutations on protein stabilization.     

Electrostatic interactions in the denatured state and in the transition state for protein folding: effects of denatured state interactions on the analysis of transition state structure

The development of electrostatic interactions during the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) is investigated by pH-dependent rate equilibrium free energy relationships. We show that Asp8, among six acidic residues, is involved in non-native, electrostatic interactions with K12 in the transition state for folding as well as in the denatured state. The perturbed native state pK(a) of D8 (pK(a) = 3.0) appears to be maintained through non-native interactions in both the transition state and the denatured state. Mutational effects on the stability of the transition state for protein (un)folding are often analyzed in respect to change in ground states. Thus, the interpretation of transition state analysis critically depends on an understanding of mutational effects on both the native and denatured state. Increasing evidence for structurally biased denatured states under physiological conditions raises concerns about possible denatured state effects on folding studies. We show that the structural interpretation of transition state analysis can be altered dramatically by denatured state effects.     

Modeling the effects of mutations on the denatured states of proteins.

We develop a model for the reversible denaturation of proteins and for the effects of single-site mutations on the denatured states. The model is based on short chains of sequences of H (hydrophobic) and P (other) monomers configured as self-avoiding walks on the two-dimensional square lattice. The N (native) state is defined as the unique conformation of lowest contact energy, whereas the D (denatured) state is defined as the collection of all other conformations. With this model we are able to determine the exact partition function, and thus the exact native-denatured equilibrium for various solvent conditions, using the computer to exhaustively enumerate every possible configuration. Previous studies confirm that this model shows many aspects of protein-like behavior. The present study attempts to model how the denatured state (1) depends on the amino acid sequence, and (2) is changed by single-site mutations. The model accounts for two puzzling experimental results: (1) the replacement of a polar residue by a hydrophobic amino acid on the surface of a protein can destabilize a native protein, and (2) the "denaturant slope," m = partial delta G/partial c (where c is the concentration of denaturant--urea, guanidine hydrochloride), can sometimes change by as much as 30% due to a single mutation. The principal conclusion of the present study is that, under strong folding conditions, the denatured conformations that are in equilibrium with the native state are not open random configurations. Instead, they are an ensemble of highly compact conformations with a distribution that depends on the residue sequence and that can be substantially altered by single mutations. Most importantly, we conclude that mutations can exert their dominant effects on protein stability by changing the entropy of folding.     

Identification of cooperative folding units in a set of native proteins.

Cooperative unfolding penalties are calculated by statistically evaluating an ensemble of denatured states derived from native structures. The ensemble of denatured states is determined by dividing the native protein into short contiguous segments and defining all possible combinations of native, i.e., interacting, and non-native, i.e., non-interacting, segments. We use a novel knowledge-based scoring function, derived from a set of non-homologous proteins in the Protein Data Bank, to describe the interactions among residues. This procedure is used for the structural identification of cooperative folding cores for four globular proteins: bovine pancreatic trypsin inhibitor, horse heart cytochrome c, French bean plastocyanin, and staphylococcal nuclease. The theoretical folding units are shown to correspond to regions that exhibit enhanced stability against denaturation as determined from experimental hydrogen exchange protection factors. Using a sequence similarity score for related sequences, we show that, in addition to residues necessary for enzymatic function, those amino acids comprising structurally important folding cores are also preferentially conserved during evolution. This implies that the identified folding cores may be part of an array of fundamental structural folding units.     

Osmolyte effects on kinetics of FKBP12 C22A folding coupled with prolyl isomerization

Unfolding and refolding kinetics of human FKBP12 C22A were monitored by fluorescence emission over a wide range of urea concentration in the presence and absence of protecting osmolytes glycerol, proline, sarcosine and trimethylamine-N-oxide (TMAO). Unfolding is well described by a mono-exponential process, while refolding required a minimum of two exponentials for an adequate fit throughout the urea concentration range considered. The bi-exponential behavior resulted from complex coupling between protein folding, and prolyl isomerization in the denatured state in which the urea-dependent rate constant for folding was greater than, equal to, and less than the rate constants for prolyl isomerization within the urea concentration range of zero to five molar. Amplitudes and the observed folding and unfolding rate constants were fitted to a reversible three-state model composed of two sequential steps involving the native state and a folding-competent denatured species thermodynamically linked to a folding-incompetent denatured species. Excellent agreement between thermodynamic parameters for FKBP12 C22A folding calculated from the kinetic parameters and those obtained directly from equilibrium denaturation assays provides strong support for the applicability of the mechanism, and provides evidence that FKBP12 C22A folding/unfolding is two-state, with prolyl isomer heterogeneity in the denatured ensemble. Despite the chemical diversity of the protecting osmolytes, they all exhibit the same kinetic behavior of increasing the rate constant of folding and decreasing the rate constant for unfolding. Osmolyte effects on folding/unfolding kinetics are readily explained in terms of principles established in understanding osmolyte effects on protein stability. These principles involve the osmophobic effect, which raises the Gibbs energy of the denatured state due to exposure of peptide backbone, thereby increasing the folding rate. This effect also plays a key role in decreasing the unfolding rate when, as is often the case, the activated complex exposes more backbone than is exposed in the native state.     

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.     

Folding pathway dependence on energetic frustration and interaction heterogeneity for a three-dimensional hydrophobic protein model

Monte Carlo simulations of a hydrophobic protein model of 40 monomers in the cubic lattice are used to explore the effect of energetic frustration and interaction heterogeneity on its folding pathway. The folding pathway is described by the dependence of relevant conformational averages on an appropriate reaction coordinate, pfold, defined as the probability for a given conformation to reach the native structure before unfolding. We compare the energetically frustrated and heterogeneous hydrophobic potential, according to which individual monomers have a higher or lower tendency to form contacts unspecifically depending on their hydrophobicities, to an unfrustrated homogeneous Go-type potential with uniformly attractive native interactions and neutral non-native interactions (called Go1 in this study), and to an unfrustrated heterogeneous potential with neutral non-native interactions and native interactions having the same energy as the hydrophobic potential (called Go2 in this study). Folding kinetics are slowed down dramatically when energetic frustration increases, as expected and previously observed in a two-dimensional model. Contrary to our previous results in two dimensions, however, it appears that the folding pathway and transition state ensemble can be significantly dependent on the energy function used to stabilize the native structure. The sequence of events along the reaction coordinate, or the order along this coordinate in which different regions of the native conformation become structured, turns out to be similar for the hydrophobic and Go2 potentials, but with analogous events tending to occur at lower pfold values in the first case. In particular, the transition state obtained from the ensemble around pfold = 0.5 is more structured for the hydrophobic potential. For Go1, not only the transition state ensemble but the order of events itself is modified, suggesting that interaction heterogeneity, in addition to energetic frustration, can have significant effects on the folding mechanism, most likely by modifying the probability of different contacts in the unfolded state, the starting point for the folding reaction. Although based on a simple model, these results provide interesting insight into how sequence-dependent switching between folding pathways might occur in real proteins.     

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.     

Electrostatic interactions in the denatured state ensemble: their effect upon protein folding and protein stability

It is now recognized that the denatured state ensemble (DSE) of proteins can contain significant amounts of structure, particularly under native conditions. Well-studied examples include small units of hydrogen bonded secondary structure, particularly helices or turns as well as hydrophobic clusters. Other types of interactions are less well characterized and it has often been assumed that electrostatic interactions play at most a minor role in the DSE. However, recent studies have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE. These can include surprisingly specific non-native interactions that can even persist in the transition state for protein folding. DSE electrostatic interactions can be energetically significant and their modulation either by mutation or by varying solution conditions can have a major impact upon protein stability. pH dependent stability studies have shown that electrostatic interactions can contribute up to 4 kcal mol^-1 to the stability of the DSE.     

Differential salt-induced stabilization of structure in the initial folding intermediate ensemble of barstar

The effects of two salts, KCl and MgCl(2), on the stability and folding kinetics of barstar have been studied at pH 8. Equilibrium urea unfolding curves were used to show that the free energy of unfolding, deltaG(UN), of barstar increased from a value of 4.7 kcalmol(-1) in the absence of salt to a value of 6.9 kcalmol(-1) in the presence of 1M KCl or 1M MgCl(2). For both salts, deltaG(UN) increases linearly with an increase in concentration of salt from 0M to 1M, suggesting that stabilization of the native state occurs primarily through a Hofmeister effect. Refolding kinetics were studied in detail in the presence of 1M KCl as well as in the presence of 1M MgCl(2), and it is shown that the basic folding mechanism is not altered upon addition of salt. The major effects on the refolding kinetics can be attributed to the stabilization of the initial burst phase ensemble, I(E), by salt. Stabilization of structure in I(E) by KCl causes the fluorescence properties of I(E) to change, so that there is an initial burst phase change in fluorescence at 320 nm, during refolding. The structure in I(E) is stabilized by MgCl(2), but no burst phase change in fluorescence at 320 nm is observed during refolding. The fluorescence emission spectra of I(E) show that when refolding is initiated in 1M KCl, the three tryptophan residues in I(E) are less solvent exposed than when folding is initiated in 1M MgCl(2). Stabilization of I(E) leads to an acceleration in the rate of the fast observable phase of folding by both salts, suggesting that structure of the transition state resembles that of I(E). The stabilization of I(E) by salts can be accounted for largely by the same mechanism that accounts for the stabilization of the native state of the protein, namely through the Hofmeister effect. The salts do not affect the rates of the slower phases of folding, indicating that the late intermediate ensemble, I(L), is not stabilized by salts. Stabilization of the native state results in deceleration of the fast unfolding rate, which has virtually no dependence on the concentration of KCl or MgCl(2) at high concentrations. The observation that the salt-induced stabilization of structure in I(E) is accompanied by an acceleration in the fast folding rate, suggests that I(E) is likely to be a productive on-pathway intermediate.     

Antibodies as specific chaperones

Protein folding is often accompanied by formation of non-native conformations leading to protein aggregation. A number of reports indicate that antibodies can facilitate folding and prevent aggregation of protein antigens. The influence of antibodies on folding is strictly antigen specific. Chaperone-like antibody activity may be due to the stabilization of native antigen conformations or folding transition states, or screening of aggregating hydrophobic surfaces. Taking advantage of chaperone-like activity of antibodies for immunotherapy may prove to be a promising approach to the treatment of Alzheimers and prion-related diseases. Antibody-assisted folding may enhance renaturation of recombinant proteins from inclusion bodies.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1515–1521.Original Russian Text Copyright © 2004 by Ermolenko, Zherdev, Dzantiev.