Salt bridge stability in monomeric proteins.

Here, we present the results of continuum electrostatic calculations on a dataset of 222 non-equivalent salt bridges derived from 36 non-homologous high-resolution monomeric protein crystal structures. Most of the salt bridges in our dataset are stabilizing, regardless of whether they are buried or exposed, isolated or networked, hydrogen bonded or non-hydrogen bonded. One-third of the salt bridges in our dataset are buried in the protein core, with the remainder exposed to the solvent. The difference in the dielectric properties of water versus the hydrophobic protein interior cost buried salt bridges large desolvation penalties. However, the electrostatic interactions both between the salt-bridging side-chains, and between the salt bridges and charges in their protein surroundings, are also stronger in the interior, due to the absence of solvent screening. Even large desolvation penalties for burying salt bridges are frequently more than compensated for, primarily by the electrostatic interactions between the salt-bridging side-chains. In networked salt bridges both types of electrostatic interactions, those between the salt-bridging side-chains, and those between the salt bridge and its protein environment, are of similar magnitudes. In particular, a major finding of this work is that salt bridge geometry is a critical factor in determining salt bridge stability. Salt bridges with favorable geometrical positioning of the interacting side-chain charged groups are likely to be stabilizing anywhere in the protein structure. We further find that most of the salt bridges are formed between residues that are relatively near each other in the sequence.     

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.     

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.     

A newly introduced salt bridge cluster improves structural and biophysical properties of de novo TIM barrels

Protein stability can be fine‐tuned by modifying different structural features such as hydrogen‐bond networks, salt bridges, hydrophobic cores, or disulfide bridges. Among these, stabilization by salt bridges is a major challenge in protein design and engineering since their stabilizing effects show a high dependence on the structural environment in the protein, and therefore are difficult to predict and model. In this work, we explore the effects on structure and stability of an introduced salt bridge cluster in the context of three different de novo TIM barrels. The salt bridge variants exhibit similar thermostability in comparison with their parental designs but important differences in the conformational stability at 25°C can be observed such as a highly stabilizing effect for two of the proteins but a destabilizing effect to the third. Analysis of the formed geometries of the salt bridge cluster in the crystal structures show either highly ordered salt bridge clusters or only single salt bridges. Rosetta modeling of the salt bridge clusters results in a good prediction of the tendency on stability changes but not the geometries observed in the three‐dimensional structures. The results show that despite the similarities in protein fold, the salt bridge clusters differently influence the structural and stability properties of the de novo TIM barrel variants depending on the structural background where they are introduced.     

Fluctuations in ion pairs and their stabilities in proteins

This report investigates the effect of systemic protein conformational flexibility on the contribution of ion pairs to protein stability. Toward this goal, we use all NMR conformer ensembles in the Protein Data Bank (1) that contain at least 40 conformers, (2) whose functional form is monomeric, (3) that are nonredundant, and (4) that are large enough. We find 11 proteins adhering to these criteria. Within these proteins, we identify 22 ion pairs that are close enough to be classified as salt bridges. These are identified in the high-resolution crystal structures of the respective proteins or in the minimized average structures (if the crystal structures are unavailable) or, if both are unavailable, in the "most representative" conformer of each of the ensembles. We next calculate the electrostatic contribution of each such ion pair in each of the conformers in the ensembles. This results in a comprehensive study of 1,201 ion pairs, which allows us to look for consistent trends in their electrostatic contributions to protein stability in large sets of conformers. We find that the contributions of ion pairs vary considerably among the conformers of each protein. The vast majority of the ion pairs interconvert between being stabilizing and destabilizing to the structure at least once in the ensembles. These fluctuations reflect the variabilities in the location of the ion pairing residues and in the geometric orientation of these residues, both with respect to each other, and with respect to other charged groups in the remainder of the protein. The higher crystallographic B-factors for the respective side-chains are consistent with these fluctuations. The major conclusion from this study is that salt bridges observed in crystal structure may break, and new salt bridges may be formed. Hence, the overall stabilizing (or, destabilizing) contribution of an ion pair is conformer population dependent.     

Contribution of salt bridges near the surface of a protein to the conformational stability

Salt bridges play important roles in the conformational stability of proteins. However, the effect of a surface salt bridge on the stability remains controversial even today; some reports have shown little contribution of a surface salt bridge to stability, whereas others have shown a favorable contribution. In this study, to elucidate the net contribution of a surface salt bridge to the conformational stability of a protein, systematic mutant human lysozymes, containing one Glu to Gln (E7Q) and five Asp to Asn mutations (D18N, D49N, D67N, D102N, and D120N) at residues where a salt bridge is formed near the surface in the wild-type structure, were examined. The thermodynamic parameters for denaturation between pH 2.0 and 4.8 were determined by use of a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. The denaturation Gibbs energy (DeltaG) of all mutant proteins was lower than that of the wild-type protein at pH 4, whereas there was little difference between them near pH 2. This is caused by the fact that the Glu and Asp residues are ionized at pH 4 but protonated at pH 2, indicating a favorable contribution of salt bridges to the wild-type structure at pH 4. Each contribution was not equivalent, but we found that the contributions correlate with the solvent inaccessibility of the salt bridges; the salt bridge contribution was small when 100% accessible, while it was about 9 kJ/mol if 100% inaccessible. This conclusion indicates how to reconcile a number of conflicting reports about role of surface salt bridges in protein stability. Furthermore, the effect of salts on surface salt bridges was also examined. In the presence of 0.2 M KCl, the stability at pH 4 decreased, and the differences in stability between the wild-type and mutant proteins were smaller than those in the absence of salts, indicating the compensation to the contribution of salt bridges with salts. Salt bridges with more than 50% accessibility did not contribute to the stability in the presence of 0.2 M KCl.     

Anticooperativity in a Glu-Lys-Glu salt bridge triplet in an isolated alpha-helical peptide

Salt bridges between oppositely charged side chains are well-known to stabilize protein structure, though their contributions vary considerably. Here we study Glu-Lys and Lys-Glu salt bridges, formed when the residues are spaced i, i + 4 surface of an isolated alpha-helix in aqueous solution. Both are stabilizing by -0.60 and -1.02 kcal/mol, respectively, when the interacting residues are fully charged. When the side chains are spaced i, i + 4, i + 8, forming a Glu-Lys-Glu triplet, the second salt bridge provides no additional stabilization to the helix. We attribute this to the inability of the central Lys to form two salt bridges simultaneously. Analysis of these salt bridges in protein structures shows that the Lys-Glu interaction is dominant, with the side chains of the Glu-Lys pair far apart.     

Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles

Coulombic interactions between charges on the surface of proteins contribute to stability. It is difficult, however, to estimate their importance by protein engineering methods because mutation of one residue in an ion pair alters the energetics of many interactions in addition to the coulombic energy between the two components. We have estimated the interaction energy between two charged residues, Asp-12 and Arg-16, in an alpha-helix on the surface of a barnase mutant by invoking a double-mutant cycle involving wild-type enzyme (Asp-12, Thr-16), the single mutants Thr----Arg-16 and Asp----Ala-12, and the double mutant Asp----Ala-12, Thr----Arg-16. The changes in free energy of unfolding of the single mutants are not additive because of the coulombic interaction energy. Additivity is restored at high concentrations of salt that shield electrostatic interactions. The geometry of the ion pair in the mutant was assumed to be the same as that in the highly homologous ribonuclease from Bacillus intermedius, binase, which has Asp-12 and Arg-16 in the native enzyme. The ion pair does not form a hydrogen-bonded salt bridge, but the charges are separated by 5-6 A. The mutant barnase containing the ion pair Asp-12/Arg-16 is more stable than wild type by 0.5 kcal/mol, but only a part of the increased stability is attributable to the electrostatic interaction. We present a formal analysis of how double-mutant cycles can be used to measure the energetics of pairwise interactions.     

The role of helix 1 aspartates and salt bridges in the stability and conversion of prion protein

A key event in the pathogenesis of transmissible spongiform encephalopathies is the conversion of PrP-sen to PrP-res. Morrissey and Shakhnovich (Morrissey, M. P., and Shakhnovich, E. I. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11293-11298) proposed that the conversion mechanism involves critical interactions at helix 1 (residues 144-153) and that the helix is stabilized on PrP-sen by intra-helix salt bridges between two aspartic acid-arginine ion pairs at positions 144 and 148 and at 147 and 151, respectively. Mutants of the hamster prion protein were constructed by replacing the aspartic acids with either asparagines or alanines to destabilize the proposed helix 1 salt bridges. Thermal and chemical denaturation experiments using circular dichroism spectroscopy indicated the overall structures of the mutants are not substantially destabilized but appear to unfold differently. Cell-free conversion reactions performed using ionic denaturants, detergents, and salts (conditions unfavorable to salt bridge formation) showed no significant differences between conversion efficiencies of mutant and wild type proteins. Using conditions more favorable to salt bridge formation, the mutant proteins converted with up to 4-fold higher efficiency than the wild type protein. Thus, although spectroscopic data indicate the salt bridges do not substantially stabilize PrP-sen, the cell-free conversion data suggest that Asp-144 and Asp-147 and their respective salt bridges stabilize PrP-sen from converting to PrP-res.     

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.     

Structural and thermodynamic studies on a salt-bridge triad in the NADP-binding domain of glutamate dehydrogenase from Thermotoga maritima: cooperativity and electrostatic contribution to stability

Cooperative interactions within ion-pair networks of hyperthermostable proteins are thought to be a major determinant for extreme protein stability. While the favorable thermodynamic contributions of optimized electrostatics in general as well as those of pairwise interactions have been documented, cooperativity between pairwise interactions has not yet been studied thermodynamically in proteins from hyperthermophiles. In this study we use the isolated cofactor binding domain of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima to analyze pairwise and cooperative interactions within the salt-bridge triad Arg190-Glu231-Lys193. The X-ray structure of the domain was solved at 1.43 A and reveals the salt-bridge network with surrounding solvent molecules in detail. All three participating charges in the network were mutated to alanine in all combinations. The X-ray structure of the variant lacking all three charges reveals that the removal of the side chains has no effect on the overall conformation of the protein. Using solvent denaturation and thermodynamic cycles, the interaction energies between each pair of residues in the network were determined in the presence and in the absence of the third residue. Both the Arg190-Glu231 ion pair and the Lys193-Glu231 salt bridge in the absence of the third residue, contribute favorably to the free energy for unfolding of the domain in urea. Using guanidinium chloride as denaturant reveals a strong cooperativity between the two ion-pair interactions, the presence of the second ion pair converts the first interaction from destabilizing into stabilizing by as much as 1.09 kcal/mol. The different energetics of the salt-bridge triad in urea and GdmCl are discussed with reference to the observed anion binding in the crystal structure at high ionic strength and their possible role in a highly charged, high-temperature environment such as the cytoplasm of hyperthermophiles.     

Peptide models of electrostatic interactions in proteins: NMR studies on two beta-turn tetrapeptides containing Asp-His and Asp-Lys salt bridges

Two model peptides Boc-Asp-Pro-Aib-X-NHMe [X = His (1) and X = Lys (2)] were synthesized to simulate intramolecular electrostatic interactions between ionizable side chains. Conformational analysis by 270-MHz 1H NMR in (CD3)2SO reveals that the backbone secondary structures of these two peptides are stabilized by two strong intramolecular hydrogen bonds, involving the consecutive carboxy-terminal NH groups. 1H NMR chemical shifts were measured in 1, 2, and a protected derivative, Boc-Asp(OBzl)-Pro-Aib-His-NHMe (3). These shifts were also measured for the model compounds Ac-Lys-NHMe, Boc-Asp-NHMe, and Boc-His-NHMe in their different states of ionization. An analysis of the chemical shifts of the ionization-sensitive reporter resonances suggests the formation of a strong intramolecular salt bridge in the lysyl peptide 2 and a bridge of moderate strength in the histidyl peptide 1. A comparison of the temperature dependence of chemical shifts in peptides 1-3 suggests that intramolecular salt bridge formation results in diminished backbone flexibility. The results establish that proximity effects confer far greater stability to intramolecular ion pair interactions vis-a-vis their intermolecular counterparts. The salt bridge interaction in peptide 1 displays a remarkable sensitivity to the dielectric constant of the solvent medium. The results suggest that these peptides are good simulators of the role of salt bridges in the structural dynamics of proteins.     

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.     

Salt bridges in the hyperthermophilic protein Ssh10b are resilient to temperature increases

A double mutant cycle (DMC) approach was employed to estimate the effect of temperature on the contribution of two highly conserved salt bridges to protein stability in the hyperthermophilic protein Ssh10b. The coupling free energy were 2.4 +/- 0.4 kJ/mol at 298 K and 2.2 +/- 0.4 kJ/mol at 353 K for Glu-54/Arg-57, and 6.0 +/- 0.2 kJ/mol at 298 K and 5.9 +/- 0.6 kJ/mol at 353 K for Glu-36/Lys-68. The stability free energy of Ssh10b decrease greatly with increasing temperature, while the direct contribution of these two salt bridges to protein stability remain almost constant, providing evidence supporting the theoretical prediction that salt bridges are extremely resilient to temperature increases and thus are specially suited to improving protein stability at high temperatures. The reason for the difference in coupling free energy between salt bridges Glu-54/Arg-57 and Glu-36/Lys-68 is discussed. Comparing our results with published DMC data for the contribution of salt bridges to stability in other proteins, we found that the energy contribution of a salt bridge formed by two charged residues far apart in the primary sequence is higher than that of those formed between two very close ones. Implications of this finding are useful for engineering proteins with enhanced thermostability.     

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.     

Contributions of engineered surface salt bridges to the stability of T4 lysozyme determined by directed mutagenesis.

Six designed mutants of T4 lysozyme were created in an attempt to create putative salt bridges on the surface of the protein. The first three of the mutants, T115E (Thr 115 to Glu), Q123E, and N144E, were designed to introduce a new charged side chain close to one or more existing charged groups of the opposite sign on the surface of the protein. In each of these cases the putative electrostatic interactions introduced by the mutation include possible salt bridges between residues within consecutive turns of an alpha-helix. Effects of the mutations ranged from no change in stability to a 1.5 degrees C (0.5 kcal/mol) increase in melting temperature. In two cases, secondary (double) mutants were constructed as controls in which the charge partner was removed from the primary mutant structure. These controls proteins indicate that the contributions to stability from each of the engineered salt bridges is very small (about 0.1-0.25 kcal/mol in 0.15 M KCl). The structures of the three primary mutants were determined by X-ray crystallography and shown to be essentially the same as the wild-type structure except at the site of the mutation. Although the introduced charges in the T115E and Q123E structures are within 3-5 A of their intended partner, the introduced side chains and their intended partners were observed to be quite mobile. It has been shown that the salt bridge between His 31 and Asp 70 in T4 lysozyme stabilizes the protein by 3-5 kcal/mol [Anderson, D. E., Becktel, W. J., & Dahlquist, F. W. (1990) Biochemistry 29, 2403-2408]. To test the effectiveness of His...Asp interactions in general, three additional double mutants, K60H/L13D, K83H/A112D, and S90H/Q122D, were created in order to introduce histidine-aspartate charge pairs on the surface of the protein. Each of these mutants destabilizes the protein by 1-3 kcal/mol in 0.15 M KCl at pH values from 2 to 6.5. The X-ray crystallographic structure of the mutant K83H/A112D has been determined and shows that there are backbone conformational changes of 0.3-0.6 A extending over several residues. The introduction of the histidine and aspartate presumably introduces strain into the folded protein that destabilizes this variant. It is concluded that pairs of oppositely charged residues that are on the surface of a protein and have freedom to adopt different conformations do not tend to come together to form structurally localized salt bridges. Rather, such residues tend to remain mobile, interact weakly if at all, and do not contribute significantly to protein stability. It is argued that the entropic cost of localizing a pair of solvent-exposed charged groups on the surface of a protein largely offsets the interaction energy expected from the formation of a defined salt bridge. There are examples of strong salt bridges in proteins, but such interactions require that the folding of the protein provides the requisite driving energy to hold the interacting partners in the correct rigid alignment.     

Salt bridges at the inter-ring interface regulate the thermostat of GroEL

The chaperonin GroEL consists of a double-ring structure made of identical subunits and displays unusual allosteric properties caused by the interaction between its constituent subunits. Cooperative binding of ATP to a protein ring allows binding of GroES to that ring, and at the same time negative inter-ring cooperativity discharges the ligands from the opposite ring, thus driving the protein-folding cycle. Biochemical and electron microscopy analysis of wild type GroEL, a single-ring mutant (SR1), and two mutants with one inter-ring salt bridge of the chaperonin disrupted (E461K and E434K) indicate that these ion pairs form part of the interactions that allow the inter-ring allosteric signal to be transmitted. The wild type-like activities of the ion pair mutants at 25 degrees C are in contrast with their lack of inter-ring communication and folding activity at physiological temperatures. These salt bridges stabilize the inter-ring interface and maintain the inter-ring spacing so that functional communication between protein heptamers takes place. The characterization of GroEL hybrids containing different amounts of wild type and mutant subunits also indicates that as the number of inter-ring salt bridges increases the functional properties of the hybrids recover. Taken together, these results strongly suggest that inter-ring salt bridges form a stabilizing ring-shaped, ionic zipper that ensures inter-ring communication at the contact sites and therefore a functional protein-folding cycle. Furthermore, they regulate the chaperonin thermostat, allowing GroEL to distinguish physiological (37 degrees C) from stress temperatures (42 degrees C).     

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.     

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.     

Effects of protein-protein interaction in ultrafiltration based fractionation processes

This paper discusses the use of pulsed sample injection ultrafiltration (UF) for investigating protein-protein interaction, particularly its effect on protein transmission through UF membranes. Several binary protein mixtures were investigated; the proteins in each mixture being selected such that one of the proteins in the pair would be preferentially transmitted while the other would be either totally or substantially retained. The "retained" protein either decreased or increased or did not affect the sieving coefficient of the "transmitted" protein, this depending the type of protein-protein interaction, that is, associative, repulsive, or neutral. The type of protein-protein interaction depended on the particular protein pair under investigation as well as on the operating conditions used (pH and salt concentration). The magnitude of either decrease or increase in transmission of a preferentially transmitted protein due to the presence of a retained protein was found to be independent of the manner in which the proteins were injected into the system, that is, simultaneous or sequential. These magnitudes however correlated well with the ratio of the two proteins present in the feed.