Mutational analysis demonstrates that specific electrostatic interactions can play a key role in the denatured state ensemble of proteins

The nature of the denatured state ensemble has been controversial for decades owing, in large part, to the difficulty in characterizing the structure and energetics of denatured state interactions. There is increasing evidence for relatively non-specific hydrophobic clustering in the denatured states of some proteins but other types of interactions are much less well characterized. Here, we report the characterization of highly specific electrostatic interactions in the denatured state of a small alpha-beta protein, the N-terminal domain of the ribosomal protein L9 (NTL9). Mutation of Lys12 to Met has been shown to increase the stability of NTL9 significantly through the disruption of denatured state interactions. Here, we describe the analysis of the pH-dependent stability of 13 mutants designed to probe the nature of the Lys12 denatured state interaction. Lys12 is located in a lysine-rich region of the protein but analysis of a set of Lys to Met mutants shows that it plays a unique role in the denatured state. Analysis of mutants of all of the acidic residues in NTL9 shows that Lys12 forms a specific non-native electrostatic interaction with Asp8 in the denatured state ensemble. Thus the distribution of charge-charge interactions in the denatured state ensemble of NTL9 appears to be biased by few key interactions and is very different from that expected in a random coil. We propose that these interactions are not encoded by local sequence effects but rather reflect interactions among residues more distant in sequence. These results demonstrate that electrostatic as well as hydrophobic interactions can play an important role in the denatured state ensemble.     

Uncovering Specific Electrostatic Interactions in the Denatured States of Proteins

The stability and folding of proteins are modulated by energetically significant interactions in the denatured state that is in equilibrium with the native state. These interactions remain largely invisible to current experimental techniques, however, due to the sparse population and conformational heterogeneity of the denatured-state ensemble under folding conditions. Molecular dynamics simulations using physics-based force fields can in principle offer atomistic details of the denatured state. However, practical applications are plagued with the lack of rigorous means to validate microscopic information and deficiencies in force fields and solvent models. This study presents a method based on coupled titration and molecular dynamics sampling of the denatured state starting from the extended sequence under native conditions. The resulting denatured-state pK_as allow for the prediction of experimental observables such as pH- and mutation-induced stability changes. I show the capability and use of the method by investigating the electrostatic interactions in the denatured states of wild-type and K12M mutant of NTL9 protein. This study shows that the major errors in electrostatics can be identified by validating the titration properties of the fragment peptides derived from the sequence of the intact protein. Consistent with experimental evidence, our simulations show a significantly depressed pK_a for Asp⁸ in the denatured state of wild-type, which is due to a nonnative interaction between Asp⁸ and Lys¹². Interestingly, the simulation also shows a nonnative interaction between Asp⁸ and Glu⁴⁸ in the denatured state of the mutant. I believe the presented method is general and can be applied to extract and validate microscopic electrostatics of the entire folding energy landscape.     

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.     

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.     

Thermodynamics of protein denatured states

Recent work on the thermodynamics of protein denatured states is providing insight into the stability of residual structure and the conformational constraints that affect the disordered states of proteins. Current data from native state hydrogen exchange and the pH dependence of protein stability indicate that residual structure can modulate the stability of the denatured state by up to 4 kcal mol(-1). NMR structural data have emphasized the role of hydrophobic clusters in stabilizing denatured state residual structures, however recent results indicate that electrostatic interactions, both favorable and unfavorable, are also important modulators of the stability of the denatured state. Thermodynamics methods that take advantage of histidine-heme ligation chemistry have also been developed to probe the conformational constraints that act on denatured states. These methods have provided insights into the role of excluded volume, chain stiffness, and loop persistence in modulating the conformational preferences of highly disordered proteins. New insights into protein folding and novel methods to manipulate protein stability are emerging from this work.     

Charge sequence coding in statistical modeling of unfolded proteins

Unfolded proteins recently attracted attention due to accumulation of experimental evidences for their significant role in different life processes. Modeling of electrostatic interactions (EI) in unfolded state of proteins is becoming increasingly important as well. In this paper, we stress on the importance of how the sequence of charged residues of a given protein is incorporated into the models for calculation of EI in the unfolded state. On the basis of the distributions of distances between titratable sites of charged residues calculated for polypeptide chains of various compositions, it was found that the distance distribution for a pair of residues, located close to each other along the sequence of a protein, depends on what residues constitute the pair in question. It was concluded that the consideration of these residue-specific distributions is essential for a statistical model to be accurate from the physical point of view. It was suggested that use of distance intervals in the spherical model of unfolded proteins accounts better for the charge sequence than the set of single distance values. This was illustrated by comparison of the pK values of the titratable groups of the unfolded N-terminal SH3 domain of the Drosophila protein drk to the available experimental data.     

pKa values and the pH dependent stability of the N-terminal domain of L9 as probes of electrostatic interactions in the denatured state. Differentiation between local and nonlocal interactions

pKa values were measured for the 6 carboxylates in the N-terminal domain of L9 (NTL9) by following NMR chemical shifts as a function of pH. The contribution of each carboxylate to the pH dependent stability of NTL9 was estimated by comparing the pKa values for the native and denatured state of the protein. A set of peptides with sequences derived from NTL9 were used to model the denatured state. In the protein fragments, the pKa values measured for the aspartates varied between 3.8 and 4.1 and the pKa values measured for the glutamates varied between 4.1 and 4.6. These results indicate that the local sequence can significantly influence pKa values in the denatured state and highlight the difficulties in using standard pKa values derived from small compounds. Calculations based on the measured pKa values suggest that the free energy of unfolding of NTL9 should decrease by 4.4 kcal mol-1 when the pH is lowered from 6 to 2. In contrast, urea and thermal denaturation experiments indicate that the stability of the protein decreases by only 2.6 kcal mol-1 when the carboxylates are protonated. This discrepancy indicates that the protein fragments are not a complete representation of the denatured state and that nonlocal sequence effects perturb the pKa's in the denatured state. Increasing the salt concentration from 100 to 750 mM NaCl removes the discrepancy between the stabilities derived from denaturation experiments and the stability changes calculated from the pKa values. At high concentrations of salt there is also less variation of the pKa values measured in the protein fragments. Our results argue that in the denatured state of NTL9 there are electrostatic interactions between groups both local and nonlocal in primary sequence.     

Electrostatic screening and backbone preferences of amino acid residues in urea-denatured ubiquitin

Local structures in denatured proteins may be important in guiding a polypeptide chain during the folding and misfolding processes. Existence of local structures in chemically denatured proteins is a highly controversial issue. NMR parameters [coupling constants (3) J(H(alpha),H(N)) and chemical shifts] of chemically denatured proteins in general deviate little from their values in small peptides. These peptides were presumed to be completely unstructured; therefore, it was considered that chemically denatured proteins are random coils. But recent experimental studies show that small peptides adopt relatively stable structures in aqueous solutions. Small deviations of the NMR parameters from their values in small peptides may thus actually indicate the existence of local structures in chemically denatured proteins. Using NMR data and theoretical predictions we show here that fluctuating beta-strands exist in urea-denatured ubiquitin (8 M urea at pH 2). Residues in such beta-strands populate more frequently the left side of the broad beta region of -psi space. Urea-denatured ubiquitin contains no detectable beta-sheet secondary structures; nevertheless, the fluctuating beta-strands in urea-denatured ubiquitin coincide to the beta-strands in the native state. Formation of beta-strands is in accord with the electrostatic screening model of unfolded proteins. The free energy of a residue in an unfolded protein is in this model determined by the local backbone electrostatics and its screening by backbone solvation. These energy terms introduce strong electrostatic coupling between neighboring residues, which causes cooperative formation of beta-strands in denatured proteins. We propose that fluctuating beta-strands in denatured proteins may serve as initiation sites to form fibrils.     

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.     

Effect of mutations involving charged residues on the stability of staphylococcal nuclease: a continuum electrostatics study

A continuum electrostatics model is used to calculate the relative stabilities of 117 mutants of staphylococcal nuclease (SNase) involving the mutation of a charged residue to an uncharged residue. The calculations are based on the crystallographic structure of the wild-type protein and attempt to take implicitly into account the effect of the mutations in the denatured state by assuming a linear relationship between the free energy changes caused by the mutation in the native and denatured states. A good correlation (linear correlation coefficient of approximately 0.8) is found with published experimental relative stabilities of these mutants. The results suggest that in the case of SNase (i) charged residues contribute to the stability of the native state mainly through electrostatic interactions, and (ii) native-like electrostatic interactions may persist in the denatured state. The continuum electrostatics method is only moderately sensitive to model parameters and leads to quasi-predictive results for the relative mutant stabilities (error of 2-3 kJ mol(-1) or of the order of k(B)T), except for mutants in which a charged residue is mutated to glycine.     

Heat capacity and compactness of denatured proteins

One of the striking results of protein thermodynamics is that the heat capacity change upon denaturation is large and positive. This change is generally ascribed to the exposure of non-polar groups to water on denaturation, in analogy to the large heat capacity change for the transfer of small non-polar molecules from hydrocarbons to water. Calculations of the heat capacity based on the exposed surface area of the completely unfolded denatured state give good agreement with experimental data. This result is difficult to reconcile with evidence that the heat denatured state in the absence of denaturants is reasonably compact. In this work, sample conformations for the denatured state of truncated CI2 are obtained by use of an effective energy function for proteins in solution. The energy function gives denatured conformations that are compact with radii of gyration that are slightly larger than that of the native state. The model is used to estimate the heat capacity, as well as that of the native state, at 300 and 350 K via finite enthalpy differences. The calculations show that the heat capacity of denaturation can have large positive contributions from non-covalent intraprotein interactions because these interactions change more with temperature in non-native conformations than in the native state. Including this contribution, which has been neglected in empirical surface area models, leads to heat capacities of unfolding for compact denatured states that are consistent with the experimental heat capacity data. Estimates of the stability curve of CI2 made with the effective energy function support the present model.     

Statistical mechanics of protein folding by cluster distance geometry

This is our second type of model for protein folding where the configurational parameters and the effective potential energy function are chosen in such a way that all conformations are described and the canonical partition function can be evaluated analytically. Structure is described in terms of distances between pairs of sequentially contiguous blocks of eight residues, and all possible conformations are grouped into 71 subsets in terms of bounds on these distances. The energy is taken to be a sum of pairwise interactions between such blocks. The 210 energy parameters were adjusted so that the native folds of 32 small proteins are favored in free energy over the denatured state. We then found 146 proteins having negligible sequence similarity to any of the training proteins, yet the free energy of the respective correct native states were favored over the denatured state.     

Coarse-grained strategy for modeling protein stability in concentrated solutions

We present a coarse-grained approach for modeling the thermodynamic stability of single-domain globular proteins in concentrated aqueous solutions. Our treatment derives effective protein-protein interactions from basic structural and energetic characteristics of the native and denatured states. These characteristics, along with the intrinsic (i.e., infinite dilution) thermodynamics of folding, are calculated from elementary sequence information using a heteropolymer collapse theory. We integrate this information into Reactive Canonical Monte Carlo simulations to investigate the connections between protein sequence hydrophobicity, protein-protein interactions, protein concentration, and the thermodynamic stability of the native state. The model predicts that sequence hydrophobicity can affect how protein concentration impacts native-state stability in solution. In particular, low hydrophobicity proteins are primarily stabilized by increases in protein concentration, whereas high hydrophobicity proteins exhibit richer nonmonotonic behavior. These trends appear qualitatively consistent with the available experimental data. Although factors such as pH, salt concentration, and protein charge are also important for protein stability, our analysis suggests that some of the nontrivial experimental trends may be driven by a competition between destabilizing hydrophobic protein-protein attractions and entropic crowding effects.     

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.     

Structured disorder and conformational selection

Traditionally, molecular disorder has been viewed as local or global instability. Molecules or regions displaying disorder have been considered inherently unstructured. The term has been routinely applied to cases for which no atomic coordinates can be derived from crystallized molecules. Yet, even when it appears that the molecules are disordered, prevailing conformations exist, with population times higher than those of all alternate conformations. Disordered molecules are the outcome of rugged energy landscapes away from the native state around the bottom of the funnel. Ruggedness has a biological function, creating a distribution of structured conformers that bind via conformational selection, driving association and multimolecular complex formation, whether chain-linked in folding or unlinked in binding. We classify disordered molecules into two types. The first type possesses a hydrophobic core. Here, even if the native conformation is unstable, it still has a large enough population time, enabling its experimental detection. In the second type, no such hydrophobic core exists. Hence, the native conformations of molecules belonging to this category have shorter population times, hindering their experimental detection. Although there is a continuum of distribution of hydrophobic cores in proteins, an empirical, statistically based hydrophobicity function may be used as a guideline for distinguishing the two disordered molecule types. Furthermore, the two types relate to steps in the protein folding reaction. With respect to protein design, this leads us to propose that engineering-optimized specific electrostatic interactions to avoid electrostatic repulsion would reduce the type I disordered state, driving the molten globule (MG) --> native (N) state. In contrast, for overcoming the type II disordered state, in addition to specific interactions, a stronger hydrophobic core is also indicated, leading to the denatured --> MG --> N state.     

Role of the charge-charge interactions in defining stability and halophilicity of the CspB proteins

Charge-charge interactions on the surface of native proteins are important for protein stability and can be computationally redesigned in a rational way to modulate protein stability. Such computational effort led to an engineered protein, CspB-TB that has the same core as the mesophilic cold shock protein CspB-Bs from Bacillus subtilis, but optimized distribution of charge-charge interactions on the surface. The CspB-TB protein shows an increase in the transition temperature by 20 degrees C relative to the unfolding temperature of CspB-Bs. The CspB-TB and CspB-Bs protein pair offers a unique opportunity to further explore the energetics of charge-charge interactions as the substitutions at the same sequence positions are done in largely similar structural but different electrostatic environments. In particular we addressed two questions. What is the contribution of charge-charge interactions in the unfolded state to the protein stability and how amino acid substitutions modulate the effect of increase in ionic strength on protein stability (i.e. protein halophilicity). To this end, we experimentally measured the stabilities of over 100 variants of CspB-TB and CspB-Bs proteins with substitutions at charged residues. We also performed computational modeling of these protein variants. Analysis of the experimental and computational data allowed us to conclude that the charge-charge interactions in the unfolded state of two model proteins CspB-Bs and CspB-TB are not very significant and computational models that are based only on the native state structure can adequately, i.e. qualitatively (stabilizing versus destabilizing) and semi-quantitatively (relative rank order), predict the effects of surface charge neutralization or reversal on protein stability. We also show that the effect of ionic strength on protein stability (protein halophilicity) appears to be mainly due to the screening of the long-range charge-charge interactions.     

De novo design of foldable proteins with smooth folding funnel: automated negative design and experimental verification

De novo sequence design of foldable proteins provides a way of investigating principles of protein architecture. We performed fully automated sequence design for a target structure having a three-helix bundle topology and synthesized the designed sequences. Our design principle is different from the conventional approach, in that instead of optimizing interactions within the target structure, we design the global shape of the protein folding funnel. This includes automated implementation of negative design by explicitly requiring higher free energy of the denatured state. The designed sequences do not have significant similarity to those of any natural proteins. The NMR and CD spectroscopic data indicated that one designed sequence has a well-defined three-dimensional structure as well as alpha-helical content consistent with the target.     

Continuum electrostatic model for the binding of cytochrome c2 to the photosynthetic reaction center from Rhodobacter sphaeroides

Electrostatic interactions are important for protein-protein association. In this study, we examined the electrostatic interactions between two proteins, cytochrome c(2) (cyt c(2)) and the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides, that function in intermolecular electron transfer in photosynthesis. Electrostatic contributions to the binding energy for the cyt c(2)-RC complex were calculated using continuum electrostatic methods based on the recent cocrystal structure [Axelrod, H. L., et al. (2002) J. Mol. Biol. 319, 501-515]. Calculated changes in binding energy due to mutations of charged interface residues agreed with experimental results for a protein dielectric constant epsilon(in) of 10. However, the electrostatic contribution to the binding energy for the complex was close to zero due to unfavorable desolvation energies that compensate for the favorable Coulomb attraction. The electrostatic energy calculated as a function of displacement of the cyt c(2) from the bound position showed a shallow minimum at a position near but displaced from the cocrystal configuration. These results show that although electrostatic steering is present, other short-range interactions must be present to contribute to the binding energy and to determine the structure of the complex. Calculations made to model the experimental data on association rates indicate a solvent-separated transition state for binding in which the cyt c(2) is displaced approximately 8 A above its position in the bound complex. These results are consistent with a two-step model for protein association: electrostatic docking of the cyt c(2) followed by desolvation to form short-range van der Waals contacts for rapid electron transfer.     

Denatured proteins and early folding intermediates simulated in a reduced conformational space

Conformations of globular proteins in the denatured state were studied using a high-resolution lattice model of proteins and Monte Carlo dynamics. The model assumes a united-atom and high-coordination lattice representation of the polypeptide conformational space. The force field of the model mimics the short-range protein-like conformational stiffness, hydrophobic interactions of the side chains and the main-chain hydrogen bonds. Two types of approximations for the short-range interactions were compared: simple statistical potentials and knowledge-based protein-specific potentials derived from the sequence-structure compatibility of short fragments of protein chains. Model proteins in the denatured state are relatively compact, although the majority of the sampled conformations are globally different from the native fold. At the same time short protein fragments are mostly native-like. Thus, the denatured state of the model proteins has several features of the molten globule state observed experimentally. Statistical potentials induce native-like conformational propensities in the denatured state, especially for the fragments located in the core of folded proteins. Knowledge-based protein-specific potentials increase only slightly the level of similarity to the native conformations, in spite of their qualitatively higher specificity in the native structures. For a few cases, where fairly accurate experimental data exist, the simulation results are in semiquantitative agreement with the physical picture revealed by the experiments. This shows that the model studied in this work could be used efficiently in computational studies of protein dynamics in the denatured state, and consequently for studies of protein folding pathways, i.e. not only for the modeling of folded structures, as it was shown in previous studies. The results of the present studies also provide a new insight into the explanation of the Levinthal's paradox.     

Electrostatic contributions to the stability of a thermophilic cold shock protein

The thermophilic Bacillus caldolyticus cold shock protein (Bc-Csp) differs from the mesophilic Bacillus subtilis cold shock protein B (Bs-CspB) in 11 of the 66 residues. Stability measurements of Schmid and co-workers have implicated contributions of electrostatic interactions to the thermostability. To further elucidate the physical basis of the difference in stability, previously developed theoretical methods that treat electrostatic effects in both the folded and the unfolded states were used in this paper to study the effects of mutations, ionic strength, and temperature. For 27 mutations that narrow the difference in sequence between Bc-Csp and Bs-CspB, calculated changes in unfolding free energy (Delta G) and experimental results have a correlation coefficient of 0.98. Bc-Csp appears to use destabilization of the unfolded state by unfavorable charge-charge interactions as a mechanism for increasing stability. Accounting for the effects of ionic strength and temperature on the electrostatic free energies in both the folded and the unfolded states, explanations for two important experimental observations are presented. The disparate ionic strength dependences of Delta G for Bc-Csp and Bs-CspB were attributed to the difference in the total charges (-2e and -6e, respectively). A main contribution to the much higher unfolding entropy of Bs-CspB was found to come from the less favorable electrostatic interactions in the folded state. These results should provide insight for understanding the thermostability of other thermophilic proteins.