Evaluation of direct and cooperative contributions towards the strength of buried hydrogen bonds and salt bridges

An experimental approach to evaluate the net binding free energy of buried hydrogen bonds and salt bridges is presented. The approach, which involves a modified multiple-mutant cycle protocol, was applied to selected interactions between TEM-1-beta-lactamase and its protein inhibitor, BLIP. The selected interactions (two salt bridges and two hydrogen bonds) all involving BLIP-D49, define a distinct binding unit. The penta mutant, where all side-chains constructing the binding unit were mutated to Ala, was used as a reference state to which combinations of side-chains were introduced. At first, pairs of interacting residues were added allowing the determination of interaction energies in the absence of neighbors, using double mutant cycles. Addition of neighboring residues allowed the evaluation of their cooperative effects on the interaction. The two isolated salt bridges were either neutral or repulsive whereas the two hydrogen bonds contribute 0.3 kcal mol(-1 )each. Conversely, a double mutant cycle analysis of these interactions in their native environment showed that they all stabilize the complex by 1-1.5 kcal mol(-1). Examination of the effects of neighboring residues on each of the interactions revealed that the formation of a salt bridge triad, which involves two connected salt bridges, had a strong cooperative effect on stabilizing the complex independent of the presence or absence of additional neighbors. These results demonstrate the importance of forming net-works of buried salt bridges. We present theoretical electrostatic calculations which predict the observed mode of cooperativity, and suggest that the cooperative networking effect results from the favorable contribution of the protein to the interaction. Furthermore, a good correlation between calculated and experimentally determined interaction energies for the two salt bridges, and to a lesser extent for the two hydrogen bonds, is shown. The data analysis was performed on values of DeltaDeltaG(double dagger)K(d) which reflect the strength of short range interactions, while DeltaDeltaG(o)K(D) values which include the effects of long range electrostatic forces that alter specifically DeltaDeltaG(double dagger)k(a) were treated separately.     

Salt bridges: geometrically specific, designable interactions

Salt bridges occur frequently in proteins, providing conformational specificity and contributing to molecular recognition and catalysis. We present a comprehensive analysis of these interactions in protein structures by surveying a large database of protein structures. Salt bridges between Asp or Glu and His, Arg, or Lys display extremely well-defined geometric preferences. Several previously observed preferences are confirmed, and others that were previously unrecognized are discovered. Salt bridges are explored for their preferences for different separations in sequence and in space, geometric preferences within proteins and at protein-protein interfaces, co-operativity in networked salt bridges, inclusion within metal-binding sites, preference for acidic electrons, apparent conformational side chain entropy reduction on formation, and degree of burial. Salt bridges occur far more frequently between residues at close than distant sequence separations, but, at close distances, there remain strong preferences for salt bridges at specific separations. Specific types of complex salt bridges, involving three or more members, are also discovered. As we observe a strong relationship between the propensity to form a salt bridge and the placement of salt-bridging residues in protein sequences, we discuss the role that salt bridges might play in kinetically influencing protein folding and thermodynamically stabilizing the native conformation. We also develop a quantitative method to select appropriate crystal structure resolution and B-factor cutoffs. Detailed knowledge of these geometric and sequence dependences should aid de novo design and prediction algorithms.     

Statistical analysis of protein structures suggests that buried ionizable residues in proteins are hydrogen bonded or form salt bridges

It is well known that nonpolar residues are largely buried in the interior of proteins, whereas polar and ionizable residues tend to be more localized on the protein surface where they are solvent exposed. Such a distribution of residues between surface and interior is well understood from a thermodynamic point: nonpolar side chains are excluded from the contact with the solvent water, whereas polar and ionizable groups have favorable interactions with the water and thus are preferred at the protein surface. However, there is an increasing amount of information suggesting that polar and ionizable residues do occur in the protein core, including at positions that have no known functional importance. This is inconsistent with the observations that dehydration of polar and in particular ionizable groups is very energetically unfavorable. To resolve this, we performed a detailed analysis of the distribution of fractional burial of polar and ionizable residues using a large set of ?2600 nonhomologous protein structures. We show that when ionizable residues are fully buried, the vast majority of them form hydrogen bonds and/or salt bridges with other polar/ionizable groups. This observation resolves an apparent contradiction: the energetic penalty of dehydration of polar/ionizable groups is paid off by favorable energy of hydrogen bonding and/or salt bridge formation in the protein interior. Our conclusion agrees well with the previous findings based on the continuum models for electrostatic interactions in proteins. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Statistical characterization of salt bridges in proteins

The structure and folding mechanism of a given protein are determined by many factors, including the electrostatic interactions between charged residues of protein molecules known in general as salt bridges. In this study, analyses were conducted on 10,370 salt bridges in 2017 proteins and the results compared to previous statistical surveys of 36 protein structures. Although many of the general trends remained consistent with other studies, more detailed information was illuminated by the larger dataset. In particular, it was shown that there is a strong correlation between secondary structure and salt bridge formation, and that salt bridges display preferential formation in an environment of about 30% solvent accessible surface area.     

Do salt bridges stabilize proteins? A continuum electrostatic analysis

The electrostatic contribution to the free energy of folding was calculated for 21 salt bridges in 9 protein X-ray crystal structures using a continuum electrostatic approach with the DELPHI computer-program package. The majority (17) were found to be electrostatically destabilizing; the average free energy change, which is analogous to mutation of salt bridging side chains to hydrophobic isosteres, was calculated to be 3.5 kcal/mol. This is fundamentally different from stability measurements using pKa shifts, which effectively measure the strength of a salt bridge relative to 1 or more charged hydrogen bonds. The calculated effect was due to a large, unfavorable desolvation contribution that was not fully compensated by favorable interactions within the salt bridge and between salt-bridge partners and other polar and charged groups in the folded protein. Some of the salt bridges were studied in further detail to determine the effect of the choice of values for atomic radii, internal protein dielectric constant, and ionic strength used in the calculations. Increased ionic strength resulted in little or no change in calculated stability for 3 of 4 salt bridges over a range of 0.1-0.9 M. The results suggest that mutation of salt bridges, particularly those that are buried, to "hydrophobic bridges" (that pack at least as well as wild type) can result in proteins with increased stability. Due to the large penalty for burying uncompensated ionizable groups, salt bridges could help to limit the number of low free energy conformations of a molecule or complex and thus play a role in determining specificity (i.e., the uniqueness of a protein fold or protein-ligand binding geometry).     

Protein stabilization by salt bridges: concepts, experimental approaches and clarification of some misunderstandings

Salt bridges in proteins are bonds between oppositely charged residues that are sufficiently close to each other to experience electrostatic attraction. They contribute to protein structure and to the specificity of interaction of proteins with other biomolecules, but in doing so they need not necessarily increase a protein's free energy of unfolding. The net electrostatic free energy of a salt bridge can be partitioned into three components: charge-charge interactions, interactions of charges with permanent dipoles, and desolvation of charges. Energetically favorable Coulombic charge-charge interaction is opposed by often unfavorable desolvation of interacting charges. As a consequence, salt bridges may destabilize the structure of the folded protein. There are two ways to estimate the free energy contribution of salt bridges by experiment: the pK(a) approach and the mutation approach. In the pK(a) approach, the contribution of charges to the free energy of unfolding of a protein is obtained from the change of pK(a) of ionizable groups caused by altered electrostatic interactions upon folding of the protein. The pK(a) approach provides the relative free energy gained or lost when ionizable groups are being charged. In the mutation approach, the coupling free energy between interacting charges is obtained from a double mutant cycle. The coupling free energy is an indirect and approximate measure of the free energy of charge-charge interaction. Neither the pK(a) approach nor the mutation approach can provide the net free energy of a salt bridge. Currently, this is obtained only by computational methods which, however, are often prone to large uncertainties due to simplifying assumptions and insufficient structural information on which calculations are based. This state of affairs makes the precise thermodynamic quantification of salt bridge energies very difficult. This review is focused on concepts and on the assessment of experimental methods and does not cover the vast literature.     

Electrostatic strengths of salt bridges in thermophilic and mesophilic glutamate dehydrogenase monomers

Here we seek to understand the higher frequency of occurrence of salt bridges in proteins from thermophiles as compared to their mesophile homologs. We focus on glutamate dehydrogenase, owing to the availability of high resolution thermophilic (from Pyrococcus furiosus) and mesophilic (from Clostridium symbiosum) protein structures, the large protein size and the large difference in melting temperatures. We investigate the location, statistics and electrostatic strengths of salt bridges and of their networks within corresponding monomers of the thermophilic and mesophilic enzymes. We find that many of the extra salt bridges which are present in the thermophilic glutamate dehydrogenase monomer but absent in the mesophilic enzyme, form around the active site of the protein. Furthermore, salt bridges in the thermostable glutamate dehydrogenase cluster within the hydrophobic folding units of the monomer, rather than between them. Computation of the electrostatic contribution of salt bridge energies by solving the Poisson equation in a continuum solvent medium, shows that the salt bridges in Pyrococcus furiosus glutamate dehydrogenase are highly stabilizing. In contrast, the salt bridges in the mesophilic Clostridium symbiosum glutamate dehydrogenase are only marginally stabilizing. This is largely the outcome of the difference in the protein environment around the salt bridges in the two proteins. The presence of a larger number of charges, and hence, of salt bridges contributes to an electrostatically more favorable protein energy term. Our results indicate that salt bridges and their networks may have an important role in resisting deformation/unfolding of the protein structure at high temperatures, particularly in critical regions such as around the active site.     

Relationship between ion pair geometries and electrostatic strengths in proteins

The electrostatic free energy contribution of an ion pair in a protein depends on two factors, geometrical orientation of the side-chain charged groups with respect to each other and the structural context of the ion pair in the protein. Conformers in NMR ensembles enable studies of the relationship between geometry and electrostatic strengths of ion pairs, because the protein structural contexts are highly similar across different conformers. We have studied this relationship using a dataset of 22 unique ion pairs in 14 NMR conformer ensembles for 11 nonhomologous proteins. In different NMR conformers, the ion pairs are classified as salt bridges, nitrogen-oxygen (N-O) bridges and longer-range ion pairs on the basis of geometrical criteria. In salt bridges, centroids of the side-chain charged groups and at least a pair of side-chain nitrogen and oxygen atoms of the ion-pairing residues are within a 4 A distance. In N-O bridges, at least a pair of the side-chain nitrogen and oxygen atoms of the ion-pairing residues are within 4 A distance, but the distance between the side-chain charged group centroids is greater than 4 A. In the longer-range ion pairs, the side-chain charged group centroids as well as the side-chain nitrogen and oxygen atoms are more than 4 A apart. Continuum electrostatic calculations indicate that most of the ion pairs have stabilizing electrostatic contributions when their side-chain charged group centroids are within 5 A distance. Hence, most (approximately 92%) of the salt bridges and a majority (68%) of the N-O bridges are stabilizing. Most (approximately 89%) of the destabilizing ion pairs are the longer-range ion pairs. In the NMR conformer ensembles, the electrostatic interaction between side-chain charged groups of the ion-pairing residues is the strongest for salt bridges, considerably weaker for N-O bridges, and the weakest for longer-range ion pairs. These results suggest empirical rules for stabilizing electrostatic interactions in proteins.     

Differences in electrostatic properties at antibody-antigen binding sites: implications for specificity and cross-reactivity

Antibodies HyHEL8, HyHEL10, and HyHEL26 (HH8, HH10, and HH26, respectively) recognize highly overlapping epitopes on hen egg-white lysozyme (HEL) with similar affinities, but with different specificities. HH8 binding to HEL is least sensitive toward mutations in the epitope and thus is most cross-reactive, HH26 is most sensitive, whereas the sensitivity of HH10 lies in between HH8 and HH26. Here we have investigated intra- and intermolecular interactions in three antibody-protein complexes: theoretical models of HH8-HEL and HH26-HEL complexes, and the x-ray crystal structure of HH10-HEL complex. Our results show that HH8-HEL has the lowest number and HH26-HEL has the highest number of intra- and intermolecular hydrogen bonds. The number of salt bridges is lowest in HH8-HEL and highest in HH26-HEL. The binding site salt bridges in HH8-HEL are not networked, and are weak, whereas, in HH26-HEL, an intramolecular salt-bridge triad at the binding site is networked to an intermolecular triad to form a pentad. The pentad and each salt bridge of this pentad are exceptionally stabilizing. The number of binding-site salt bridges and their strengths are intermediate in HH10-HEL, with an intramolecular triad. Our further calculations show that the electrostatic component contributes the most to binding energy of HH26-HEL, whereas the hydrophobic component contributes the most in the case of HH8-HEL. A "hot-spot" epitope residue Lys-97 forms an intermolecular salt bridge in HH8-HEL, and participates in the intermolecular pentad in the HH26-HEL complex. Mutant modeling and surface plasmon resonance (SPR) studies show that this hot-spot epitope residue contributes significantly more to the binding than an adjacent epitope residue, Lys-96, which does not form a salt bridge in any of the three HH-HEL complexes. Furthermore, the effect of mutating Lys-97 is most severe in HH26-HEL. Lys-96, being a charged residue, also contributes the most in HH26-HEL among the three complexes. The SPR results on these mutants also highlight that the apparent "electrostatic steering" on net on rates actually act at post-collision level stabilization of the complex. The significance of this work is the observed variations in electrostatic interactions among the three complexes. Our work demonstrates that higher electrostatics, both as a number of short-range electrostatic interactions and their contributions, leads to higher binding specificity. Strong salt bridges, their networking, and electrostatically driven binding, limit flexibilities through geometric constrains. In contrast, hydrophobic driven binding and low levels of electrostatic interactions are associated with conformational flexibility and cross-reactivity.     

Surface salt bridges stabilize the GCN4 leucine zipper.

We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcal/ mol, an apparent elevation of the melting temperature relative to wild type of about 22 degrees C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences.     

On the role of electrostatic interactions in the design of protein-protein interfaces

Here, the methods of continuum electrostatics are used to investigate the contribution of electrostatic interactions to the binding of four protein-protein complexes; barnase-barstar, human growth hormone and its receptor, subtype N9 influenza virus neuraminidase and the NC41 antibody, the Ras binding domain (RBD) of kinase cRaf and a Ras homologue Rap1A. In two of the four complexes electrostatics are found to strongly oppose binding (hormone-receptor and neuraminidase-antibody complexes), in one case the net effect is close to zero (barnase-barstar) and in one case electrostatics provides a significant driving force favoring binding (RBD-Rap1A). In order to help understand the wide range of electrostatic contributions that were calculated, the electrostatic free energy was partitioned into contributions of individual charged and polar residues, salt bridges and networks involving salt bridges and hydrogen bonds. Although there is no one structural feature that accounts for the differences between the four interfaces, the extent to which the desolvation of buried charges is compensated by the formation of hydrogen bonds and ion pairs appears to be an important factor. Structural features that are correlated with contribution of an individual residue to stability are also discussed. These include partial burial of a charged group in the free monomer, the formation of networks involving charged and polar amino acids, and the formation of partially exposed ion-pairs. The total electrostatic contribution to binding is found to be inversely correlated with buried total and non-polar surface area. This suggests that different interfaces can be designed to exploit electrostatic and hydrophobic forces in very different ways.     

Strength and co-operativity of contributions of surface salt bridges to protein stability

Many of the interactions that stabilize proteins are co-operative and cannot be reduced to a sum of pairwise interactions. Such interactions may be analysed by protein engineering methods using multiple thermodynamic cycles comprising wild-type protein and all combinations of mutants in the interacting residues. There is a triad of charged residues on the surface of barnase, comprising residues Asp8, Asp12 and Arg110, that interact by forming two exposed salt bridges. The three residues have been mutated to alanine to give all the single, double and triple mutants. The free energies of unfolding of wild-type and the seven mutant proteins have been determined and the results analysed to give the contributions of the residues in the two salt bridges to protein stability. It is possible to isolate the energies of forming the salt bridges relative to the solvation of the separated ions by water. In the intact triad, the apparent contribution to the stabilization energy of the protein of the salt bridge between Asp12 and Arg110 is -1.25 kcal mol-1, whereas that of the salt bridge between Asp8 with Arg110 is -0.98 kcal mol-1. The strengths of the two salt bridges are coupled: the energy of each is reduced by 0.77 kcal mol-1 when the other is absent. The salt-linked triad, relative to alanine residues at the same positions, does not contribute to the stability of the protein since the favourable interactions of the salt bridges are more than offset by other electrostatic and non-electrostatic energy terms. Salt-linked triads occur in other proteins, for example, haemoglobin, where the energy of only the salt-bridge term is important and so the coupling of salt bridges could be of general importance to the stability and function of proteins.     

Cooperative helix stabilization by complex Arg-Glu salt bridges

Among the interactions that stabilize the native state of proteins, the role of electrostatic interactions has been difficult to quantify precisely. Surface salt bridges or ion pairs between acidic and basic side chains have only a modest stabilizing effect on the stability of helical peptides or proteins: estimates are roughly 0.5 kcal/mol or less. On the other hand, theoretical arguments and the occurrence of salt bridge networks in thermophilic proteins suggest that multiple salt bridges may exert a stronger stabilizing effect. We show here that triads of charged side chains, Arg(+)-Glu(-)-Arg(+) spaced at i,i+4 or i,i+3 intervals in a helical peptide stabilize alpha helix by more than the additive contribution of two single salt bridges. The free energy of the triad is more than 1 kcal/mol in excess of the sum of the individual pairs, measured in low salt concentration (10 mM). The effect of spacing the three groups is severe; placing the charges at i,i+4 or i,i+3 sites has a strong effect on stability relative to single bridges; other combinations are weaker. A conservative calculation suggests that interactions of this kind between salt bridges can account for much of the stabilization of certain thermophilic proteins.     

The crystal structure of the Bacillus lentus alkaline protease, subtilisin BL, at 1.4 A resolution.

The crystal structure of subtilisin BL, an alkaline protease from Bacillus lentus with activity at pH 11, has been determined to 1.4 A resolution. The structure was solved by molecular replacement starting with the 2.1 A structure of subtilisin BPN' followed by molecular dynamics refinement using X-PLOR. A final crystallographic R-factor of 19% overall was obtained. The enzyme possesses stability at high pH, which is a result of the high pI of the protein. Almost all of the acidic side-chains are involved in some type of electrostatic interaction (ion pairs, calcium binding, etc.). Furthermore, three of seven tyrosine residues have potential partners for forming salt bridges. All of the potential partners are arginine with a pK around 12. Lysine would not function well in a salt bridge with tyrosine as it deprotonates at around the same pH as tyrosine ionizes. Stability at high pH is acquired in part from the pI of the protein, but also from the formation of salt bridges (which would affect the pI). The overall structure of the enzyme is very similar to other subtilisins and shows that the subtilisin fold is more highly conserved than would be expected from the differences in amino acid sequence. The amino acid side-chains in the hydrophobic core are not conserved, though the inter-residue interactions are. Finally, one third of the serine side-chains in the protein have multiple conformations. This presents an opportunity to correlate computer simulations with observed occupancies in the crystal structure.     

Effects of salt bridges on protein structure and design.

Theoretical calculations (Hendsch ZS & Tidor B, 1994, Protein Sci 3:211-226) and experiments (Waldburger CD et al., 1995, Nat Struct Biol 2:122-128; Wimley WC et al., 1996, Proc Natl Acad Sci USA 93:2985-2990) suggest that hydrophobic interactions are more stabilizing than salt bridges in protein folding. The lack of apparent stability benefit for many salt bridges requires an alternative explanation for their occurrence within proteins. To examine the effect of salt bridges on protein structure and stability in more detail, we have developed an energy function for simple cubic lattice polymers based on continuum electrostatic calculations of a representative selection of salt bridges found in known protein crystal structures. There are only three types of residues in the model, with charges of -1, 0, or + 1. We have exhaustively enumerated conformational space and significant regions of sequence space for three-dimensional cubic lattice polymers of length 16. The results demonstrate that, while the more highly charged sequences are less stable, the loss of stability is accompanied by a substantial reduction in the degeneracy of the lowest-energy state. Moreover, the reduction in degeneracy is greater due to charges that pair than for lone charges that remain relatively exposed to solvent. We have also explored and illustrated the use of ion-pairing strategies for rational structural design using model lattice studies.     

Electrostatic contributions to T4 lysozyme stability: solvent-exposed charges versus semi-buried salt bridges

We carried our Poisson-Boltzmann (PB) calculations for the effects of charge reversal at five exposed sites (K16E, R119E, K135E, K147E, and R154E) and charge neutralization and proton titration of the H31-D70 semi-buried salt bridge on the stability of T4 lysozyme. Instead of the widely used solvent-exclusion (SE) surface, we used the van der Waals (vdW) surface as the boundary between the protein and solvent dielectrics (a protocol established in our earlier study on charge mutations in barnase). By including residual charge-charge interactions in the unfolded state, the five charge reversal mutations were found to have DeltaDeltaG(unfold) from -1.6 to 1.3 kcal/mol. This indicates that the variable effects of charge reversal observed by Matthews and co-workers are not unexpected. The H31N, D70N, and H31N/D70N mutations were found to destabilize the protein by 2.9, 1.3, and 1.6 kcal/mol, and the pK(a) values of H31 and D70 were shifted to 9.4 and 0.6, respectively. These results are in good accord with experimental data of Dahlquist and co-workers. In contrast, if the SE surface were used, the H31N/D70N mutant would be more stable than the wild-type protein by 1.3 kcal/mol. From these and additional results for 27 charge mutations on five other proteins, we conclude that 1) the popular view that electrostatic interactions are generally destabilizing may have been based on overestimated desolvation cost as a result of using the SE surface as the dielectric boundary; and 2) while solvent-exposed charges may not reliably contribute to protein stability, semi-buried salt bridges can provide significant stabilization.     

Computation of electrostatic complements to proteins: a case of charge stabilized binding.

Recent evidence suggests that the net effect of electrostatics is generally to destabilize protein binding due to large desolvation penalties. A novel method for computing ligand-charge distributions that optimize the tradeoff between ligand desolvation penalty and favorable interactions with a binding site has been applied to a model for barnase. The result is a ligand-charge distribution with a favorable electrostatic contribution to binding due, in part, to ligand point charges whose direct interaction with the binding site is unfavorable, but which make strong intra-molecular interactions that are uncloaked on binding and thus act to lessen the ligand desolvation penalty.     

Acidic groups docked to well defined wetted pockets at the core of the binding interface: a tale of scoring and missing protein interactions in CAPRI

Some challenging targets in CAPRI (T24/25 and T26) involve binding solvent accessible acidic residues at the core of the binding interface, where they are always found immersed in crystal waters. In fact, Asp and Glu residues are more likely to form part of the hydrogen bond network of their surrounding crystal water molecules than to form a buried salt bridge. Interestingly, many of the crystal waters mediating the intermolecular interactions of the acidic groups are already present in the unbound structure, reinforcing the notion that some water molecules behave as an extension of the protein structure. This is in contrast to acidic groups found in the periphery of the binding interface that form ubiquitous salt bridges that cement the high affinity complex, while at the same time they are exposed to rapidly exchanging water molecules. Because of this, dichotomy implicit solvent scoring functions fail to properly rank these complexes by prioritizing salt bridges rather than water mediated contacts. A detailed analysis of Target 24, for which our group predicted two out of the four successful homology model complex structures, and Target 26 reveal how crystal waters shape the binding cavities of acidic groups prior to binding, in agreement with the theory of anchor residues as mediators of protein recognition.     

Salt-Bridge Energetics in Halophilic Proteins

Halophilic proteins have greater abundance of acidic over basic and very low bulky hydrophobic residues. Classical electrostatic stabilization was suggested as the key determinant for halophilic adaptation of protein. However, contribution of specific electrostatic interactions (i.e. salt-bridges) to overall stability of halophilic proteins is yet to be understood. To understand this, we use Adaptive-Poison-Boltzmann-Solver Methods along with our home-built automation to workout net as well as associated component energy terms such as desolvation energy, bridge energy and background energy for 275 salt-bridges from 20 extremely halophilic proteins. We then perform extensive statistical analysis on general and energetic attributes on these salt-bridges. On average, 8 salt-bridges per 150 residues protein were observed which is almost twice than earlier report. Overall contributions of salt-bridges are −3.0 kcal mol⁻¹. Majority (78%) of salt-bridges in our dataset are stable and conserved in nature. Although, average contributions of component energy terms are equal, their individual details vary greatly from one another indicating their sensitivity to local micro-environment. Notably, 35% of salt-bridges in our database are buried and stable. Greater desolvation penalty of these buried salt-bridges are counteracted by stable network salt-bridges apart from favorable equal contributions of bridge and background terms. Recruitment of extensive network salt-bridges (46%) with a net contribution of −5.0 kcal mol⁻¹ per salt-bridge, seems to be a halophilic design wherein favorable average contribution of background term (−10 kcal mol⁻¹) exceeds than that of bridge term (−7 kcal mol⁻¹). Interiors of proteins from halophiles are seen to possess relatively higher abundance of charge and polar side chains than that of mesophiles which seems to be satisfied by cooperative network salt-bridges. Overall, our theoretical analyses provide insight into halophilic signature in its specific electrostatic interactions which we hope would help in protein engineering and bioinformatics studies.     

Surface electrostatic interactions contribute little of stability of barnase

Electrostatic interactions are believed to play an important role in stabilizing the native structure of proteins. We have quantified the contribution to stability of an interaction between two oppositely charged side-chains on the surface of barnase. Using site-directed mutagenesis, glutamate 28 and lysine 32 were introduced onto the solvent-accessible side of the second alpha-helix in barnase. These two residues are separated by one turn of the helix, and so are ideally situated for their opposite charges to interact. Double mutant cycle analysis reveals that the interaction between Glu28 and Lys32 contributes only approximately 0.2 kcal/mol to stability of the protein. All other interactions between exposed charged side-chains in barnase examined so far also contribute little to stability. We explain this low value by their location on the surface, rather than in the interior, of the protein.