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.     

The folding pathway of a functionally competent C-terminal domain of nucleophosmin: Protein stability and denatured state residual structure

Nucleophosmin (NPM1) is a nucleolar protein implicated in ribosome biogenesis, centrosome duplication and cell cycle control; the NPM1 gene is the most frequent target for mutations in Acute Myeloid Leukemia. Mutations map to the C-terminal domain of the protein and cause its unfolding, loss of DNA binding properties and aberrant cellular localization. Here we investigate the folding pathway and denatured state properties of a NPM1 C-terminal domain construct encompassing the last 70 residues in the reference sequence. This construct is more stable than the previously characterized domain, which consisted of the last 53 residues. Data reveal that, similarly to what was discovered for the shorter construct, also the 70-residue construct of NPM1 displays a detectable residual structure in its denatured state. The higher stability of the latter domain allows us to conclude that the denatured state is robust to changes in solvent composition and that it consists of a discrete state in equilibrium with the expanded fully unfolded conformation. This observation, which might appear as a technicality, is in fact of general importance for the understanding of the folding of proteins. The implications of our results are discussed in the context of previous works on single domain helical proteins.     

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.     

Perturbations of the denatured state ensemble: modeling their effects on protein stability and folding kinetics.

By considering the denatured state of a protein as an ensemble of conformations with varying numbers of sequence-specific interactions, the effects on stability, folding kinetics, and aggregation of perturbing these interactions can be predicted from changes in the molecular partition function. From general considerations, the following conclusions are drawn: (1) A perturbation that enhances a native interaction in denatured state conformations always increases the stability of the native state. (2) A perturbation that promotes a non-native interaction in the denatured state always decreases the stability of the native state. (3) A change in the denatured state ensemble can alter the kinetics of aggregation and folding. (4) The loss (or increase) in stability accompanying two mutations, each of which lowers (or raises) the free energy of the denatured state, will be less than the sum of the effects of the single mutations, except in cases where both mutations affect the same set of partially folded conformations. By modeling the denatured state as the ensemble of all non-native conformations of hydrophobic-polar (HP) chains configured on a square lattice, it can be shown that the stabilization obtained from enhancement of native interactions derives in large measure from the avoidance of non-native interactions in the D state. In addition, the kinetic effects of fixing single native contacts in the denatured state or imposing linear gradients in the HH contact probabilities are found, for some sequences, to significantly enhance the efficiency of folding by a simple hydrophobic zippering algorithm. Again, the dominant mechanism appears to be avoidance of non-native interactions. These results suggest stabilization of native interactions and imposition of gradients in the stability of local structure are two plausible mechanisms involving the denatured state that could play a role in the evolution of protein folding and stability.     

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.     

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.     

pH-dependent stability and folding kinetics of a protein with an unusual alpha-beta topology: the C-terminal domain of the ribosomal protein L9

The folding kinetics and thermodynamics of the isolated C-terminal domain of the ribosomal protein L9 (CTL9) have been studied as a function of pH. CTL9 is an alpha-beta protein that contains a single beta-sheet with an unusual mixed parallel, anti-parallel topology. The folding is fully reversible and two-state over the entire pH range. Stopped-flow fluorescence and CD experiments yield the same folding rate, and the chevron plots have the characteristic V-shape expected for two-state folding. The values of DeltaG*(H2O) and the m value calculated from the kinetic experiments are in excellent agreement with the equilibrium measurements. The extrapolated initial amplitudes of both the stopped-flow fluorescence and CD measurements show that there is no detectable burst phase intermediate. The domain contains three histidine residues, two of which are largely buried in the native state. They do not participate in salt-bridges or take part in a hydrogen bonded network. NMR measurements reveal that the buried histidine residues have significantly perturbed pK(a) values in the native state. The equilibrium stability and the folding rate are found to be strongly dependent upon their ionization state. There is a linear relationship between the log of the folding rate and DeltaG* (H2O) . The protein is much more stable and folds noticeably faster at pH values above the native state pK(a) values. DeltaG*(H2O) of unfolding increases from 2.90 kcal mol(-1) at pH 5.0 to 6.40 kcal mol(-1) at pH 8.0 while the folding rate increases from 0.60 to 18.7 s(-1). Tanford linkage analysis revealed that the interactions involving the two histidine residues are largely developed in the transition state. The results are compared to other studies of the pH-dependence of folding.     

NMR and SAXS characterization of the denatured state of the chemotactic protein CheY: implications for protein folding initiation

The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X-ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies show that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexible links. A comparison of the contour length obtained for the chain at 5 M urea with the one expected for a fully expanded chain suggests that approximately 25% of the residues are involved in residual structures. Conformational shifts of the alpha-protons, heteronuclear (15)N-[(1)H] NOEs and (15)N relaxation properties have been used to identify some regions in the protein that deviate from a random coil behavior. According to these NMR data, the protein can be divided into two subdomains, which largely coincide with the two folding subunits identified in a previous kinetic study of the folding of the protein. The first of these subdomains, spanning residues 1-70, is shown here to exhibit a restricted mobility as compared to the rest of the protein. Two regions, one in each subdomain, were identified as deviating from the random coil chemical shifts. Peptides corresponding to these sequences were characterized by NMR and their backbone (1)H chemical shifts were compared to those in the intact protein under identical denaturing conditions. For the region located in the first subdomain, this comparison shows that the observed deviation from random coil parameters is caused by interactions with the rest of the molecule. The restricted flexibility of the first subdomain and the transient collapse detected in that subunit are consistent with the conclusions obtained by applying the protein engineering method to the characterization of the folding reaction transition state.     

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.     

Effect of surface electrostatic interactions on the stability and folding of formate dehydrogenase from Candida methylica

NAD⁺-dependent formate dehydrogenase (FDH-EC 1.2.1.2) is an important enzyme to regenerate valuable NADH required by NAD⁺-dependent oxidoreductases in enzyme catalysis. The limitation in the thermostability of FDH enzyme is a crucial problem for development of biotechnological and industrial processes, despite of its advantages. In this study, to investigate the contribution of surface electrostatic interaction to the thermostability of FDH from Candida methylica (cmFDH) N187E, H13E, Q105R, N300E, N147R N300E/N147R, N187E/Q105R, N187E/N147R,Y160R, Y302R, Y160E and Y302E mutants were designed using a homology model of cmFDH based on Candida boidinii (cb) by considering electrostatic interactions on the protein surface. The effects of site-specific engineering on the stability of this molecule was analyzed according to minimal model of folding and assembly reaction and deduced equilibrium properties of the native system with respect to its thermal and denaturant sensitivities. It was observed that mutations did not change the unfolding pattern of native cmFDH and increased numbers of electrostatic interactions can cause either stabilizing or destabilizing effect on the thermostability of this protein. The thermodynamic and kinetic results suggested that except relatively improved mutants, three out of the nine single mutations increased the melting temperature of cmFDH enzyme.     

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.     

Rapid cooperative two-state folding of a miniature alpha-beta protein and design of a thermostable variant

There is a great deal of interest in developing small stably folded miniature proteins. A limited number of these molecules have been described, however they typically have not been characterized in depth. In particular, almost no detailed studies of the thermodynamics and folding kinetics of these proteins have been reported. Here we describe detailed studies of the thermodynamics and kinetics of folding of a 39 residue mixed alpha-beta protein (NTL9(1-39)) derived from the N-terminal domain of the ribosomal protein L9. The protein folds cooperatively and rapidly in a two-state fashion to a native state typical of those found for normal globular proteins. At pH 5.4 in 20mM sodium acetate, 100mM NaCl the temperature of maximum stability is 6 degrees C, the t(m) is 65.3 degrees C, deltaH degrees (t(m)) is between 24.6 kcalmol(-1) and 26.3 kcalmol(-1), and deltaC(p) degrees is 0.38 kcalmol(-1)deg(-1). The thermodynamic parameters are in the range expected on the basis of per residue values determined from databases of globular proteins. H/2H exchange measurements reveal a set of amides that exchange via global unfolding, exactly as expected for a normal cooperatively folded globular protein. Kinetic measurements show that folding is two-state folding. The folding rate is 640 s(-1) and the value of deltaG degrees calculated from the folding and unfolding rates is in excellent agreement with the equilibrium value. A designed thermostable variant, generated by mutating K12 to M, was characterized and found to have a t(m) of 82 degrees C. Equilibrium and kinetic measurements demonstrate that its folding is cooperative and two-state.     

Inter-residue interactions in protein folding and stability

During the process of protein folding, the amino acid residues along the polypeptide chain interact with each other in a cooperative manner to form the stable native structure. The knowledge about inter-residue interactions in protein structures is very helpful to understand the mechanism of protein folding and stability. In this review, we introduce the classification of inter-residue interactions into short, medium and long range based on a simple geometric approach. The features of these interactions in different structural classes of globular and membrane proteins, and in various folds have been delineated. The development of contact potentials and the application of inter-residue contacts for predicting the structural class and secondary structures of globular proteins, solvent accessibility, fold recognition and ab initio tertiary structure prediction have been evaluated. Further, the relationship between inter-residue contacts and protein-folding rates has been highlighted. Moreover, the importance of inter-residue interactions in protein-folding kinetics and for understanding the stability of proteins has been discussed. In essence, the information gained from the studies on inter-residue interactions provides valuable insights for understanding protein folding and de novo protein design.     

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.     

Increasing protein stability: Importance of ΔCp and the denatured state

Increasing the conformational stability of proteins is an important goal for both basic research and industrial applications. In vitro selection has been used successfully to increase protein stability, but more often site‐directed mutagenesis is used to optimize the various forces that contribute to protein stability. In previous studies, we showed that improving electrostatic interactions on the protein surface and improving the β‐turn sequences were good general strategies for increasing protein stability, and used them to increase the stability of RNase Sa. By incorporating seven of these mutations in RNase Sa, we increased the stability by 5.3 kcal/mol. Adding one more mutation, D79F, gave a total increase in stability of 7.7 kcal/mol, and a melting temperature 28°C higher than the wild‐type enzyme. Surprisingly, the D79F mutation lowers the change in heat capacity for folding, ΔC_p, by 0.6 kcal/mol/K. This suggests that this mutation stabilizes structure in the denatured state ensemble. We made other mutants that give some insight into the structure present in the denatured state. Finally, the thermodynamics of folding of these stabilized variants of RNase Sa are compared with those observed for proteins from thermophiles.     

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.     

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.     

Amide proton exchange measurements as a probe of the stability and dynamics of the N-terminal domain of the ribosomal protein L9: comparison with the intact protein.

Amide H/D exchange rates have been measured for the N-terminal domain of the ribosomal protein L9, residues 1-56. The rates were measured at pD 3.91, 5.03, and 5.37. At pD 5.37, 18 amides exchange slowly enough to give reliable rate measurements. At pD 3.91, seven additional residues could be followed. The exchange is shown to occur by the EX2 mechanism for all conditions studied. The rates for the N-terminal domain are very similar to those previously measured for the corresponding region in the full-length protein (Lillemoen J et al., 1997, J Mol Biol 268:482-493). In particular, the rates for the residues that we have shown to exchange via global unfolding in the N-terminal domain agree within the experimental error with the rates measured by Hoffman and coworkers, suggesting that the structure of the domain is preserved in isolation and that the stability of the isolated domain is comparable to the stability of this domain in intact L9.     

Commitment and nucleation in the protein G transition state

An accurate characterization of the transition state ensemble (TSE) is central to furthering our understanding of the protein folding reaction. We have extensively tested a recently reported method for studying a protein's TSE, utilizing phi-value data from protein engineering experiments and computational studies as restraints in all-atom Monte Carlo (MC) simulations. The validity of interpreting experimental phi-values as the fraction of native contacts made by a residue in the TSE was explored, revealing that this definition is unable to uniquely specify a TSE. The identification of protein G's second hairpin, in both pre and post-transition conformations demonstrates that high experimental phi-values do not guarantee a residue's importance in the TSE. An analysis of simulations based on structures restrained by experimental phi-values is necessary to yield this result, which is not obvious from a simplistic interpretation of individual phi-values. The TSE that we obtain corresponds to a single, specific nucleation event, characterized by six residues common to all three observed, convergent folding pathways. The same specific nucleus was independently identified from computational and experimental data, and "Conservation of Conservation" analysis in the protein G fold. When associated strictly with complete nucleus formation and concomitant chain collapse, folding is a well-defined two state event. Once the nucleus has formed, the folding reaction enters a slow relaxation process associated with side-chain packing and small, local backbone rearrangements. A detailed analysis of phi-values and their relationship to the transition state ensemble allows us to construct a unified theoretical model of protein G folding.