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.     

Effects of charge-to-alanine substitutions on the stability of ribosomal protein L30e from Thermococcus celer

The ability to rationally engineer a protein with altered stability depends upon the detailed understanding of the role of noncovalent interactions in defining thermodynamic properties of proteins. In this paper, we used T. celer L30e as a model to address the question of the role of charge-charge interactions in defining the stability of this protein. A total of 26 single-site charge-to-alanine variants of this protein were generated, and the stability of these proteins was determined using thermal- and denaturant-induced unfolding. It was found that, although L30e is isolated from a thermophilic organism and is highly thermostable, some of the substitutions lead to a further increase in the transition temperature. Analysis of the effects of high ionic strength on the stabilities of L30e variants shows that the long-range charge-charge interactions are as important as the short-range (salt bridge) interactions. The changes in stabilities of the T. celer L30e protein variants were compared with the changes in the energy of charge-charge interactions calculated using different computational models. It was found that there is a good qualitative agreement between experimental and calculated data: for 70-80% (19-21 of 26, confidence p < 0.003) of the variants, computational models predict correctly the sign of the stability changes. In particular, computational models identify correctly those charged amino acid residue substitutions of which led to enhancement in thermostability. Thus, optimization of the charge-charge interactions might be a useful approach for the rational increase in protein stability.     

Contribution of surface salt bridges to protein stability: guidelines for protein engineering

The small globular protein, ubiquitin, contains a pair of oppositely charged residues, K11 and E34, that according to the three-dimensional structure are located on the surface of this protein with a spatial orientation characteristic of a salt bridge. We investigated the strength of this salt bridge and its contribution to the global stability of the ubiquitin molecule. Using the "double mutant cycle" analysis, the strength of the pairwise interactions between K11 and E34 was estimated to be favorable by 3.6kJ/mol. Further, the salt bridge of the reverse orientation, i.e. E11/K34, can be formed and is found to have a strength (3.8kJ/mol) similar to that of the K11/E34 pair. However, the global stability of the K11/E34 variant of ubiquitin is 2.2kJ/mol higher than that of the E11/K34 variant. The difference in the contribution of the opposing salt bridge orientations to the overall stability of the ubiquitin molecule is attributed to the difference in the charge-charge interactions between residues forming the salt bridge and the rest of the ionizable groups in this protein. On the basis of these results, we concluded that surface salt bridges are stabilizing, but their contribution to the overall protein stability is strongly context-dependent, with charge-charge interactions being the largest determinant. Analysis of 16 salt bridges from six different proteins, for which detailed experimental data on energetics have been reported, support the conclusions made from the analysis of the salt bridge in ubiquitin. Implications of these findings for engineering proteins with enhanced thermostability are discussed.     

Computational design of the Fyn SH3 domain with increased stability through optimization of surface charge charge interactions

Computational design of surface charge-charge interactions has been demonstrated to be an effective way to increase both the thermostability and the stability of proteins. To test the robustness of this approach for proteins with predominantly beta-sheet secondary structure, the chicken isoform of the Fyn SH3 domain was used as a model system. Computational analysis of the optimal distribution of surface charges showed that the increase in favorable energy per substitution begins to level off at five substitutions; hence, the designed Fyn sequence contained four charge reversals at existing charged positions and one introduction of a new charge. Three additional variants were also constructed to explore stepwise contributions of these substitutions to Fyn stability. The thermodynamic stabilities of the variants were experimentally characterized using differential scanning calorimetry and far-UV circular dichroism spectroscopy and are in very good agreement with theoretical predictions from the model. The designed sequence was found to have increased the melting temperature, DeltaT (m) = 12.3 +/- 0.2 degrees C, and stability, DeltaDeltaG(25 degrees C) = 7.1 +/- 2.2 kJ/mol, relative to the wild-type protein. The experimental data suggest that a significant increase in stability can be achieved through a very small number of amino acid substitutions. Consistent with a number of recent studies, the presented results clearly argue for a seminal role of surface charge-charge interactions in determining protein stability and suggest that the optimization of surface interactions can be an attractive strategy to complement algorithms optimizing interactions in the protein core to further enhance protein stability.     

Energetics of charge-charge interactions between residues adjacent in sequence

Statistical analysis of the residue separation between a pair of ionizable side chains within 4 ? of each other was performed on a set of 1560 non-homologous PDB structures. We found that the frequency of pairs of like charges (i.e., pairs consisting of acidic residues Asp and Glu or pairs consisting of basic residues Arg and Lys) is two orders of magnitude lower than the pairs of oppositely charged residues (salt-bridges). We also found that for pairs of like charges the distribution is skewed dramatically towards short residue separation (<3). On the basis of these observations, we hypothesize that at short residue separation the repulsion between charges does not contribute much to the protein stability and the effects are largely dominated by the long range charge-charge interactions with other ionizable groups in the protein molecule. To test this hypothesis, we incorporated various pairs of charged residues at position 63 and 64 of ubiquitin and compared the stabilities of these variants. We also performed calculations of the expected changes in the charge-charge interactions. A very good correlation between experimental changes in the stability of ubiquitin variants, and changes in the energy of charge-charge interactions provides support for the hypothesis that a pair of ionizable residues next to each other in sequence modulates protein stability via long range charge-charge interactions with the rest of the protein.     

Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability.

We have characterized the guanidine-induced unfolding of both yeast and bovine ubiquitin at 25 degrees C and in the acidic pH range on the basis of fluorescence and circular dichroism measurements. Unfolding Gibbs energy changes calculated by linear extrapolation from high guanidine unfolding data are found to depend very weakly on pH. A simple explanation for this result involves the two following assumptions: (1) charged atoms of ionizable groups are exposed to the solvent in native ubiquitin (as supported by accessible surface area calculations), and Gibbs energy contributions associated with charge desolvation upon folding (a source of pK shifts) are small; (2) charge-charge interactions (another source of pK shifts upon folding) are screened out in concentrated guanidinium chloride solutions. We have also characterized the thermal unfolding of both proteins using differential scanning calorimetry. Unfolding Gibbs energy changes calculated from the calorimetric data do depend strongly on pH, a result that we attribute to the pH dependence of charge-charge interactions (not eliminated in the absence of guanidine). In fact, we find good agreement between the difference between the two series of experimental unfolding Gibbs energy changes (determined from high guanidine unfolding data by linear extrapolation and from thermal denaturation data in the absence of guanidine) and the theoretical estimates of the contribution from charge-charge interactions to the Gibbs energy change for ubiquitin unfolding obtained by using the solvent-accessibility-corrected Tanford-Kirkwood model, together with the Bashford-Karplus (reduced-set-of-sites) approximation. This contribution is found to be stabilizing at neutral pH, because most charged groups on the native protein interact mainly with groups of the opposite charge, a fact that, together with the absence of large charge-desolvation contributions, may explain the high stability of ubiquitin at neutral pH. In general, our analysis suggests the possibility of enhancing protein thermal stability by adequately redesigning the distribution of solvent-exposed, charged residues on the native protein surface.     

Geometric cooperativity and anticooperativity of three-body interactions in native proteins

Characterizing multibody interactions of hydrophobic, polar, and ionizable residues in protein is important for understanding the stability of protein structures. We introduce a geometric model for quantifying 3-body interactions in native proteins. With this model, empirical propensity values for many types of 3-body interactions can be reliably estimated from a database of native protein structures, despite the overwhelming presence of pairwise contacts. In addition, we define a nonadditive coefficient that characterizes cooperativity and anticooperativity of residue interactions in native proteins by measuring the deviation of 3-body interactions from 3 independent pairwise interactions. It compares the 3-body propensity value from what would be expected if only pairwise interactions were considered, and highlights the distinction of propensity and cooperativity of 3-body interaction. Based on the geometric model, and what can be inferred from statistical analysis of such a model, we find that hydrophobic interactions and hydrogen-bonding interactions make nonadditive contributions to protein stability, but the nonadditive nature depends on whether such interactions are located in the protein interior or on the protein surface. When located in the interior, many hydrophobic interactions such as those involving alkyl residues are anticooperative. Salt-bridge and regular hydrogen-bonding interactions, such as those involving ionizable residues and polar residues, are cooperative. When located on the protein surface, these salt-bridge and regular hydrogen-bonding interactions are anticooperative, and hydrophobic interactions involving alkyl residues become cooperative. We show with examples that incorporating 3-body interactions improves discrimination of protein native structures against decoy conformations. In addition, analysis of cooperative 3-body interaction may reveal spatial motifs that can suggest specific protein functions.     

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.     

Kinetic consequences of native state optimization of surface-exposed electrostatic interactions in the Fyn SH3 domain

Optimization of surface exposed charge-charge interactions in the native state has emerged as an effective means to enhance protein stability; but the effect of electrostatic interactions on the kinetics of protein folding is not well understood. To investigate the kinetic consequences of surface charge optimization, we characterized the folding kinetics of a Fyn SH3 domain variant containing five amino acid substitutions that was computationally designed to optimize surface charge-charge interactions. Our results demonstrate that this optimized Fyn SH3 domain is stabilized primarily through an eight-fold acceleration in the folding rate. Analyses of the constituent single amino acid substitutions indicate that the effects of optimization of charge-charge interactions on folding rate are additive. This is in contrast to the trend seen in folded state stability, and suggests that electrostatic interactions are less specific in the transition state compared to the folded state. Simulations of the transition state using a coarse-grained chain model show that native electrostatic contacts are weakly formed, thereby making the transition state conducive to nonspecific, or even nonnative, electrostatic interactions. Because folding from the unfolded state to the folding transition state for small proteins is accompanied by an increase in charge density, nonspecific electrostatic interactions, that is, generic charge density effects can have a significant contribution to the kinetics of protein folding. Thus, the interpretation of the effects of amino acid substitutions at surface charged positions may be complicated and consideration of only native-state interactions may fail to provide an adequate picture.     

Protein stability and electrostatic interactions between solvent exposed charged side chains

To investigate the contribution to protein stability of electrostatic interactions between charged surface residues, we have studied the effect of substituting three negatively charged solvent exposed residues with their side-chain amide analogs in bovine calbindin D9k--a small (Mr 8,500) globular protein of the calmodulin superfamily. The free energy of urea-induced unfolding for the wild-type and seven mutant proteins has been measured. The mutant proteins have increased stability towards unfolding relative to the wild-type. The experimental results correlate reasonably well with theoretically calculated relative free energies of unfolding and show that electrostatic interactions between charges on the surface of a protein can have significant effects on protein stability.     

Removal of surface charge-charge interactions from ubiquitin leaves the protein folded and very stable

The contribution of solvent-exposed charged residues to protein stability was evaluated using ubiquitin as a model protein. We combined site-directed mutagenesis and specific chemical modifications to first replace all Arg residues with Lys, followed by carbomylation of Lys-amino groups. Under the conditions in which all carboxylic groups are protonated (at pH 2), the chemically modified protein is folded and very stable (DeltaG = 18 kJ/mol). These results indicate that surface charge-charge interactions are not an essential fundamental force for protein folding and stability.     

Investigations on the role of π–π interactions and π–π networks in eNOS and nNOS proteins

π–π Interactions play an important role in the stability of protein structures. In the present study, we have analyzed the influence of π–π interactions in eNOS and nNOS proteins. The contribution of these π–π interacting residues in sequential separation, secondary structure involvement, solvent accessibility and stabilization centers has been evaluated. π–π interactions stabilize the core regions within eNOS and nNOS proteins. π–π interacting residues are evolutionary conserved. There is a significant number of π–π interactions in spite of the lesser natural occurrences of π-residues in eNOS and nNOS proteins. In addition to π–π interactions, π residues also form π–π networks in both eNOS and nNOS proteins which might play an important role in the structural stability of these protein structures.     

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.     

Significant role of electrostatic interactions for stabilization of protein assemblies

Contribution of electrostatic interactions to stability of BPTI orthorhombic, pig-insulin cubic crystals, and horse L ferritin crystals was evaluated with numerical calculation of Poisson-Boltzmann equation based on a dielectric model. The stability of a ferritin molecule (24-mer) composed of 24 subunits was also evaluated. It was found that the surface charge-charge interactions at separation distances (< 5 Å) were insensitive to variations in the ionic strength, and thus stabilized assembled states of the proteins (i.e., crystalline state and oligomeric state). It was also revealed that the charge density and the packing of the protein crystals were largely responsible for the ionic strength dependence of the crystal stability. The stability of the 5PTI crystalline state with a high charge density drastically increased as the concentration of the solvent ions increased. In contrast, that of the insulin crystal with a low charge density and large solvent region was insensitive to changes in the ionic concentration. The electrostatic interaction between ferritin 24-mers was attributed to two salt bridges mediated by Cd ion. For the stability of the ferritin 24-mer, which is evolutionally designed, the electrostatic stabilization between the subunits was attributed to polar bonds such as buried salt bridges or hydrogen bonds, which occasionally yielded more than 5 kcal/mol and were numerous and very strong compared with the bonds between molecules in the 5PTI and 9INS crystals.By analyzing the atomic charge-charge interactions in detail, it was found that charge pairs separated by less than 3 Å, such as hydrogen bonds, dominantly stabilize the assembled states, and that pairs 3 to 5 Å apart were also important. The stability of the assembled states evaluated by the total EET was determined by the fine balance between the two competing contributions arising from the stabilizing atoms and the destabilizing atoms.Changes of the ASA and hydration free energy were also evaluated in accordance with the process of the subunit assembly. The change of hydration free energy, which was very large (i.e., ~+ 100 kcal/mol/subunit) and unfavorable for the assembly, was proportional to the electrostatic hydration energy (i.e., Born energy change in the hydration process). Hydrophobic groups were likely to appear more frequently than hydrophilic groups at the interfaces.This study offers a method which can improve the stability of protein crystals by introducing polar or charged residues that are properly designed to form specific hydrogen bonds or salt bridges between neighboring protein molecules. This method is also applicable to crystallography, because it improves refinement of protein structures in crystals by taking the inter-protein interactions into account.     

Optimization of the electrostatic interactions in proteins of different functional and folding type

The 3-dimensional optimization of the electrostatic interactions between the charged amino acid residues was studied by Monte Carlo simulations on an extended representative set of 141 protein structures with known atomic coordinates. The proteins were classified by different functional and structural criteria, and the optimization of the electrostatic interactions was analyzed. The optimization parameters were obtained by comparison of the contribution of charge-charge interactions to the free energy of the native protein structures and for a large number of randomly distributed charge constellations obtained by the Monte Carlo technique. On the basis of the results obtained, one can conclude that the charge-charge interactions are better optimized in the enzymes than in the proteins without enzymatic functions. Proteins that belong to the mixed αβ folding type are electrostatically better optimized than pure α-helical or β-strand structures. Proteins that are stabilized by disulfide bonds show a lower degree of electrostatic optimization. The electrostatic interactions in a native protein are effectively optimized by rejection of the conformers that lead to repulsive charge-charge interactions. Particularly, the rejection of the repulsive contacts seems to be a major goal in the protein folding process. The dependence of the optimization parameters on the choice of the potential function was tested. The majority of the potential functions gave practically identical results.     

The efficiency of different salts to screen charge interactions in proteins: a Hofmeister effect?

Understanding the screening by salts of charge-charge interactions in proteins is important for at least two reasons: a), screening by intracellular salt concentration may modulate the stability and interactions of proteins in vivo; and b), the in vitro experimental estimation of the contributions from charge-charge interactions to molecular processes involving proteins is generally carried out on the basis of the salt effect on process energetics, under the assumption that these interactions are screened out by moderate salt concentrations. Here, we explore experimentally the extent to which the screening efficiency depends on the nature of the salt. To this end, we have carried out an energetic characterization of the effect of NaCl (a nondenaturing salt), guanidinium chloride (a denaturing salt), and guanidinium thiocyanate (a stronger denaturant) on the stability of the wild-type form and a T14K variant of Escherichia coli thioredoxin. Our results suggest that the efficiency of different salts to screen charge-charge interactions correlates with their denaturing strength and with the position of the constituent ions in the Hofmeister rankings. This result appears consistent with the plausible relation of the Hofmeister rankings with the extent of solute accumulation/exclusion from protein surfaces.     

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.     

The effect of protein relaxation on charge-charge interactions and dielectric constants of proteins.

The effect of the reorganization of the protein polar groups on charge-charge interaction and the corresponding effective dielectric constant (epsilon(eff)) is examined by the semimicroscopic version of the Protein Dipole Langevin Dipoles (PDLD/S) method within the framework of the Linear Response Approximation (LRA). This is done by evaluating the interactions between ionized residues in the reaction center of Rhodobacter sphaeroides, while taking into account the protein reorganization energy. It is found that an explicit consideration of the protein relaxation leads to a significant increase in epsilon(eff) and that semimicroscopic models that do not take this relaxation into account force one to use a large value for the so-called "protein dielectric constant," epsilon(p), of the Poisson-Boltzmann model or for the corresponding epsilon(in) in the PDLD/S model. An additional increase in epsilon(eff) is expected from the reorganization of ionized residues and from changes in the degree of water penetration. This finding provides further support for the idea that epsilon(in) (or epsilon(p)) represents contributions that are not considered explicitly. The present study also provides a systematic illustration of the nature of epsilon(eff), supporting our previously reported view that charge-charge interactions correspond to a large value of this "dielectric constant," even in protein interiors. It is also pointed out that epsilon(eff) for the interaction between ionizable groups in proteins is very different from the effective dielectric constant, epsilon'(eff), that determines the free energy of ion pairs in proteins (epsilon'(eff) reflects the effect of preoriented protein dipoles). Finally, the problems associated with the search for a general epsilon(in) are discussed. It is clarified that the epsilon(in) that reproduces the effect of protein relaxation on charge-charge interaction is not equal to the epsilon(in) that reproduces the corresponding effect upon formation of individual charges. This reflects fundamental inconsistencies in attempts to cast microscopic concepts in a macroscopic model. Thus one should either use a large epsilon(in) for charge-charge interactions and a small epsilon(in) for charge-dipole interactions or consider the protein relaxation microscopically.     

Structural insights into the evolution of a non-biological protein: importance of surface residues in protein fold optimization

Phylogenetic profiling of amino acid substitution patterns in proteins has led many to conclude that most structural information is carried by interior core residues that are solvent inaccessible. This conclusion is based on the observation that buried residues generally tolerate only conserved sequence changes, while surface residues allow more diverse chemical substitutions. This notion is now changing as it has become apparent that both core and surface residues play important roles in protein folding and stability. Unfortunately, the ability to identify specific mutations that will lead to enhanced stability remains a challenging problem. Here we discuss two mutations that emerged from an in vitro selection experiment designed to improve the folding stability of a non-biological ATP binding protein. These mutations alter two solvent accessible residues, and dramatically enhance the expression, solubility, thermal stability, and ligand binding affinity of the protein. The significance of both mutations was investigated individually and together, and the X-ray crystal structures of the parent sequence and double mutant protein were solved to a resolution limit of 2.8 and 1.65 A, respectively. Comparative structural analysis of the evolved protein to proteins found in nature reveals that our non-biological protein evolved certain structural features shared by many thermophilic proteins. This experimental result suggests that protein fold optimization by in vitro selection offers a viable approach to generating stable variants of many naturally occurring proteins whose structures and functions are otherwise difficult to study.