Asp79 makes a large, unfavorable contribution to the stability of RNase Sa

The two most buried carboxyl groups in ribonuclease Sa (RNase Sa) are Asp33 (99% buried; pK 2.4) and Asp79 (85% buried; pK 7.4). Above these pK values, the stability of the D33A variant is 6kcal/mol less than wild-type RNase Sa, and the stability of the D79A variant is 3.3kcal/mol greater than wild-type RNase Sa. The key structural difference between the carboxyl groups is that Asp33 forms three intramolecular hydrogen bonds, and Asp79 forms no intramolecular hydrogen bond. Here, we focus on Asp79 and describe studies of 11 Asp79 variants. Most of the variants were at least 2kcal/mol more stable than wild-type RNase Sa, and the most interesting was D79F. At pH 3, below the pK of Asp79, RNase Sa is 0.3kcal/mol more stable than the D79F variant. At pH 8.5, above the pK of Asp79, RNase Sa is 3.7kcal/mol less stable than the D79F variant. The unfavorable contribution of Asp79 to the stability appears to result from the Born self-energy of burying the charge and, more importantly, from unfavorable charge-charge interactions. To counteract the effect of the negative charge on Asp79, we prepared the Q94K variant and the crystal structure showed that the amino group of the Lys formed a hydrogen-bonded ion pair (distance, 2.71A; angle, 100 degrees ) with the carboxyl group of Asp79. The stability of the Q94K variant was about the same as the wild-type at pH 3, where Asp79 is uncharged, but 1kcal/mol greater than that of wild-type RNase Sa at pH 8.5, where Asp79 is charged. Differences in hydrophobicity, steric strain, Born self-energy, and electrostatic interactions all appear to contribute to the range of stabilities observed in the variants. When it is possible, replacing buried, non-hydrogen bonded, ionizable side-chains with non-polar side-chains is an excellent means of increasing protein stability.     

Contributions of a hydrogen bond/salt bridge network to the stability of secondary and tertiary structure in lambda repressor.

In the N-terminal domain of lambda repressor, the Asp 14 side chain forms an intrahelical, hydrogen bond/salt bridge with the Arg 17 side chain and a tertiary hydrogen bond with the Ser 77 side chain. By measuring the stabilities to urea denaturation of the wild-type N-terminal domain and variants containing single, double, and triple alanine substitutions at positions 14, 17, and 77, the side-chain interaction energies, the coupling energy between interactions, and the intrinsic effects of each wild-type side chain on protein stability have been estimated. These studies indicate that the Asp 14-Arg 17 and Asp 14-Ser 77 interactions are stabilizing by roughly 0.8 and 1.5 kcal/mol, respectively, but that Asp 14, by itself, is destabilizing by roughly 0.9 kcal/mol. We also show that a peptide model of alpha-helix 1, which contains Asp 14 and Arg 17, forms a reasonably stable, monomeric helix in solution and responds to alanine mutations at positions 14 and 17 in the fashion expected from the intact protein studies. These studies suggest that it is possible to view the stability effects of mutations in intact proteins in a hierarchical fashion, with the stability of units of secondary structure being distinguishable from the stability of tertiary structure.     

Buried, charged, non-ion-paired aspartic acid 76 contributes favorably to the conformational stability of ribonuclease T1.

The side-chain carboxyl of Asp 76 in ribonuclease T1 (RNase T1) is buried, charged, non-ion-paired, and forms three good intramolecular hydrogen bonds (2.63, 2.69, and 2.89 A) and a 2.66 A hydrogen bond to a buried, conserved water molecule. When Asp 76 was replaced by Asn, Ser, and Ala, the conformational stability of the protein decreased by 3.1, 3.2, and 3.7 kcal/mol, respectively. The stability was measured as a function of pH for wild-type RNase T1 and the D76N mutant and showed that the pH dependence below pH 3 was almost entirely due to Asp 76. The pK of Asp 76 is 0.5 in the native state and 3.7 in the denatured state. Thus, the hydrogen bonding of the carboxyl group of Asp 76 contributes more than half of the net stability of RNase T1 at pH 7. In addition, the charged carboxyl of Asp 76 stabilizes structure in the denatured states of RNase T1 that is not present in D76N, D76S, and D76A.     

Influence of key residues on the reaction mechanism of the cAMP-dependent protein kinase

The reaction mechanism of the catalytic phosphoryl transfer of cAMP-dependent protein kinase (cAPK) was investigated by semi-empirical AM1 molecular orbital computations of an active site model system derived from the crystal structure of the catalytic subunit of the enzyme. The activation barrier is calculated as 20.7 kcal mol(-1) and the reaction itself to be exothermic by 12.2 kcal mol(-1). The active site residue Asp166, which was often proposed to act as a catalytic base, does not accept a proton in any of the reaction steps. Instead, the hydroxyl hydrogen of serine is shifted to the simultaneously transferred phosphate group of ATP. Although the calculated transition state geometry indicates an associative phosphoryl transfer, no concentration of negative charge is found. To study the influence of protein mutations on the reaction mechanism, we compared two-dimensional energy hypersurfaces of the protein kinase wild-type model and a corresponding mutant in which Asp166 was replaced by alanine. Surprisingly, they show similar energy profiles despite the experimentally known decrease of catalytic activity for corresponding mutants. Furthermore, a model structure was examined, where the charged NH3 group of Lys168 was replaced by a neutral methyl group. The energetic hypersurface of this hypothetical mutant shows two possible pathways for phosphoryl transfer, which both require significantly higher activation energies than the other systems investigated, while the energetic stabilization of the reaction product is similar in all systems. As the position of the amino acid side chains and the substrate peptide is virtually unchanged in all model systems, our results suggest that the exchange of Asp166 by other amino acid is less important to the phosphoryl transfer itself, but crucial to maintain the configuration of the active site in vivo. The positively charged side chain of Lys168, however, is necessary to stabilize the intermediate reaction states, particularly the side chain of the substrate peptide.     

Importance of hydrogen-bonding interactions involving the side chain of Asp158 in the catalytic mechanism of papain

In a previous study, it was shown that replacing Asp158 in papain by Asn had little effect on activity and that the negatively charged carboxylate of Asp158 does not significantly stabilize the active site thiolate-imidazolium ion pair of papain (Ménard et al., 1990). In this paper, we report the kinetic characterization of three more mutants at this position: Asp158Gly, Asp158Ala, and Asp158Glu. From the pH-activity profiles of these and other mutants of papain, it has been possible to develop a model that enables us to dissect out the contribution of the various mutations toward (i) intrinsic activity, (ii) ion pair stability, and (iii) the electrostatic potential at the active site. Results obtained with mutants that place either Gly or Ala at position 158 indicate that the hydrogen bonds involving the side chain of Asp158 in wild-type papain are indirectly important for enzyme activity. When CBZ-Phe-Arg-MCA is used as a substrate, the (kcat/KM)obs values at pH 6.5 are 3650 and 494 M-1 s-1 for Asp158Gly and Asp158Ala, respectively, as compared to 119,000 M-1 s-1 for papain. Results with the Asp158Glu mutant suggest that the side chain of Glu moves closer to the active site and cannot form hydrogen bonds similar to those involving Asp158 in papain. From the four mutations introduced at position 158 in papain, we can conclude that it is not the charge but the hydrogen-bonding interactions involving the side chain of Asp158 that contribute the most to the stabilization of the thiolate-imidazolium ion pair in papain. However, the charge and the hydrogen bonds of Asp158 both contribute to the intrinsic activity of the enzyme.     

Cumulative site-directed charge-change replacements in bacteriophage T4 lysozyme suggest that long-range electrostatic interactions contribute little to protein stability

Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9.0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby alpha-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituent single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

The PD...(D/E)XK motif in restriction enzymes: a link between function and conformation

The active sites of Mg(II)-dependent nucleases feature a cluster of conserved charged residues which includes both acidic (Asp and Glu) and basic (Lys) side chains. In restriction enzymes, these side chains are part of the conserved PD...(D/E)XK functional sequence motif which has been implicated as being important in metal ion binding and catalytic steps. Recent work revealing the unusual behavior of the active site variant D58A of the representative PvuII endonuclease prompted speculation that the array of charged groups in the nuclease active site may also be linked to conformational behavior [Dupureur, C. M., and Conlan, L. H. (2000) Biochemistry 39, 10921-10927]. To address this issue, we analyzed the conformational behavior of active site variants of PvuII endonuclease using both NMR spectroscopic and thermodynamic methods. NMR spectroscopic analysis via (19)F and (1)H-(15)N HSQC experiments indicates that a number of side chain and backbone amide groups are perturbed upon Ala substitution at conserved active site residues Asp58, Glu68, and Lys70. Spectral changes are particularly pronounced for the lowest-activity mutants (D58A and K70A). These changes are accompanied by perturbations in conformational stability. Ala substitution at each of these positions results in 2-5 kcal/mol of stabilization over the wild-type enzyme at pH 7.7, changes which constitute increases in DeltaG(d)(H2O) of 20-50%. The pH dependencies of mutant enzyme stabilities are distinct from those of the wild type, results which confirm that these ionizable groups strongly influence stability. Wild-type enzyme stability is correlated with the ionization of groups shown to be important to metal ion binding and orientation. Correlations between spectral changes and conformational stability indicate that the latter measurements may prove useful in the evaluation of site-directed mutant restriction enzymes. More importantly, these results indicate that structure-function relationships in restriction enzyme active sites can be complex, and that the ensemble of conserved charged residues which mediate DNA hydrolysis in Mg(II)-dependent nucleases constitutes a critical link between function and conformation.     

The conserved, buried aspartic acid in oxidized Escherichia coli thioredoxin has a pKa of 7.5. Its titration produces a related shift in global stability.

Aspartic acid 26 in Escherichia coli thioredoxin is located at the bottom of a hydrophobic cavity, near the redox-active disulfide of the active site. Asp 26 is embedded in the protein except for part of the surface of one carboxyl oxygen. The high degree of evolutionary conversion of Asp 26 suggests that it plays a critical role in thioredoxin function. We have determined the pKa of Asp 26 by a novel electrophoretic method based on the relative electrophoretic mobilities of wild-type thioredoxin and of D26A thioredoxin (with Asp 26 replaced by alanine). The pKa of Asp 26 determined by this technique is 7.5, more than 3 units above the pKa of a solvated carboxyl side chain. The titration of Asp 26 is thermodynamically linked to the stability of thioredoxin. As expected if thioredoxin stability depends on the ionization state of Asp 26, delta Go WT, the free energy of the cooperative denaturation reaction of wild-type thioredoxin by guanidine hydrochloride, varies with pH in a sigmoidal fashion in the vicinity of pH 7.5. Over the same pH range, the free energy for D26A folding, delta Go D26A, is pH independent and D26A is highly stabilized compared to wild type. From the thermodynamic cycle describing the linkage of Asp 26 titration to thioredoxin stability, the difference in free energy between wild-type thioredoxin with protonated Asp 26 and wild-type thioredoxin with deprotonated Asp 26, delta delta Go (COOH----COO-), is calculated to be 4.9 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Hydrophobic core repacking and aromatic-aromatic interaction in the thermostable mutant of T4 lysozyme Ser 117-->Phe.

The T4 lysozyme mutant Ser 117-->Phe was isolated fortuitously and found to be more thermostable than wild-type by 1.1-1.4 kcal/mol. In the wild-type structure, the side chain of Ser 117 is in a sterically restricted region near the protein surface and forms a short hydrogen bond with Asn 132. The crystal structure of the S117F mutant shows that the introduced Phe side chain rotates by about 150 degrees about the C alpha-C beta bond relative to wild type and is buried in the hydrophobic core of the protein. Burial of Phe 117 is accommodated by rearrangements of the surrounding side chains of Leu 121, Leu 133, and Phe 153 and by main-chain shifts, which result in a minimal increase in packing density. The benzyl rings of Phe 117 and Phe 153 form a near-optimal edge-face interaction in the mutant structure. This aromatic-aromatic interaction, as well as increased hydrophobic stabilization and elimination of a close contact in the wild-type protein, apparently compensate for the loss of a hydrogen bond and the possible cost of structural rearrangements in the mutant. The structure illustrates the ability of a protein to accommodate a surprisingly large structural change in a manner that actually increases thermal stability. The mutant has activity about 10% that of wild-type, supportive of the prior hypothesis (Grütter, M.G. & Matthews, B.W., 1982, J. Mol. Biol. 154, 525-535) that the peptidoglycan substrate of T4 lysozyme makes extended contacts with the C-terminal domain in the vicinity of Ser 117.     

Reversal of negative charges on the surface of Escherichia coli thioredoxin: pockets versus protrusions

Recent studies of proteins with reversed charged residues have demonstrated that electrostatic interactions on the surface can contribute significantly to protein stability. We have used the approach of reversing negatively charged residues using Arg to evaluate the effect of the electrostatics context on the transition temperature (T(m)), the unfolding Gibbs free energy change (DeltaG), and the unfolding enthalpy change (DeltaH). We have reversed negatively charged residues at a pocket (Asp9) and protrusions (Asp10, Asp20, Glu85), all located in interconnecting segments between elements of secondary structure on the surface of Arg73Ala Escherichia coli thioredoxin. DSC measurements indicate that reversal of Asp in a pocket (Asp9Arg/Arg73Ala, DeltaT(m) = -7.3 degrees C) produces a larger effect in thermal stability than reversal at protrusions: Asp10Arg/Arg73Ala, DeltaT(m) = -3.1 degrees C, Asp20Arg/Arg73Ala, DeltaT(m) = 2.0 degrees C, Glu85Arg/Arg73Ala, DeltaT(m) = 3.9 degrees ). The 3D structure of thioredoxin indicates that Asp20 and Glu85 have no nearby charges within 8 A, while Asp9 does not only have Asp10 as sequential neighbor, but it also forms a 5-A long-range ion pair with the solvent-exposed Lys69. Further DSC measurements indicate that neutralization of the individual charges of the ion pair Asp9-Lys69 with nonpolar residues produces a significant decrease in stability in both cases: Asp9Ala/Arg73Ala, DeltaT(m) = -3.7 degrees C, Asp9Met/Arg73Ala, DeltaT(m) = -5.5 degrees C, Lys69Leu/Arg73Ala, DeltaT(m) = -5.1 degrees C. However, thermodynamic analysis shows that reversal or neutralization of Asp9 produces a 9-15% decrease in DeltaH, while both reversal of Asp at protrusions and neutralization of Lys69 produce negligible changes. These results correlate well with the NMR analysis, which demonstrates that only the substitution of Asp9 produces extensive conformational changes and these changes occur in the surroundings of Lys69. Our results led us to suggest that reversal of a negative charge at a pocket has a larger effect on stability than a similar reversal at a protrusion and that this difference arises largely from short-range interactions with polar groups within the pocket, rather than long-range interactions with solvent-exposed charged groups.     

Substitution of aspartic acid with glutamic acid increases the unfolding transition temperature of a protein

Proteins from thermophiles are more stable than those from mesophiles. Several factors have been suggested as causes for this greater stability, but no general rule has been found. The amino acid composition of thermophile proteins indicates that the content of polar amino acids such as Asn, Gln, Ser, and Thr is lower, and that of charged amino acids such as Arg, Glu, and Lys is higher than in mesophile proteins. Among charged amino acids, however, the content of Asp is even lower in thermophile proteins than in mesophile proteins. To investigate the reasons for the lower occurrence of Asp compared to Glu in thermophile proteins, Glu was substituted with Asp in a hyperthermophile protein, MjTRX, and Asp was substituted with Glu in a mesophile protein, ETRX. Each substitution of Glu with Asp decreased the Tm of MjTRX by about 2 degrees C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 degrees C. The change of Tm destabilizes the MjTRX by 0.55 kcal/mol and stabilizes the ETRX by 0.45 kcal/mol in free energy.     

Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408-->Glu/Lys939-->Arg mutant retained significant activity, while an Asp407-->Glu/Lys939-->Arg mutant exhibited only marginal function. An Asp407-->Glu/Asp408-->Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407-->Glu/Asp408-->Glu/Lys939-->Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.     

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Asp102. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-N zeta is solvent inaccessible and is less than 5 A from the catalytic Asp102. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kcat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.     

Crystal and molecular structure of human plasminogen kringle 4 refined at 1.9-A resolution.

The crystal structure of human plasminogen kringle 4 (PGK4) has been solved by molecular replacement using the bovine prothrombin kringle 1 (PTK1) structure as a model and refined by restrained least-squares methods to an R factor of 14.2% at 1.9-A resolution. The K4 structure is similar to that of PTK1, and an insertion of one residue at position 59 of the latter has minimal effect on the protein folding. The PGK4 structure is highly stabilized by an internal hydrophobic core and an extensive hydrogen-bonding network. Features new to this kringle include a cis peptide bond at Pro30 and the presence of two alternate, perpendicular, and equally occupied orientations for the Cys75 side chain. The K4 lysine-binding site consists of a hydrophobic trough formed by the Trp62 and Trp72 indole rings, with anionic (Asp55/Asp57) and cationic (Lys35/Arg71) charge pairs at either end. With the adjacent Asp5 and Arg32 residues, these result in triply charged anionic and cationic clusters (pH of crystals at 6.0), which, in addition to the unusually high accessibility of the Trp72 side chain, serve as an obvious marker of the binding site on the K4 surface. A complex intermolecular interaction occurs between the binding sites of symmetry-related molecules involving a highly ordered sulfate anion of solvation in which the Arg32 side chain of a neighboring kringle occupies the binding site.     

Factors contributing to decreased protein stability when aspartic acid residues are in beta-sheet regions

Asp residues are significantly under represented in beta-sheet regions of proteins, especially in the middle of beta-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a beta-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V(L)) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a beta-strand, and that of Q89D, located in the middle of a beta-strand, reduced the stability of the parent immunoglobulin V(L) domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V(L)-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V(L)-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V(L)-Len domain. The structures of mutants V(L)-Len Q38D and V(L)-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these Q-->D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a beta-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a beta-strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo.     

Nine hydrophobic side chains are key determinants of the thermodynamic stability and oligomerization status of tumour suppressor p53 tetramerization domain.

The contribution of almost each amino acid side chain to the thermodynamic stability of the tetramerization domain (residues 326-353) of human p53 has been quantitated using 25 mutants with single-residue truncations to alanine (or glycine). Truncation of either Leu344 or Leu348 buried at the tetramer interface, but not of any other residue, led to the formation of dimers of moderate stability (8-9 kcal/mol of dimer) instead of tetramers. One-third of the substitutions were moderately destabilizing (<3.9 kcal/mol of tetramer). Truncations of Arg333, Asn345 or Glu349 involved in intermonomer hydrogen bonds, Ala347 at the tetramer interface or Thr329 were more destabilizing (4.1-5.7 kcal/mol). Strongly destabilizing (8.8- 11.7 kcal/mol) substitutions included those of Met340 at the tetramer interface and Phe328, Arg337 and Phe338 involved peripherally in the hydrophobic core. Truncation of any of the three residues involved centrally in the hydrophobic core of each primary dimer either prevented folding (Ile332) or allowed folding only at high protein concentration or low temperature (Leu330 and Phe341). Nine hydrophobic residues per monomer constitute critical determinants for the stability and oligomerization status of this p53 domain.     

Stabilization of a fibronectin type III domain by the removal of unfavorable electrostatic interactions on the protein surface

It is generally considered that electrostatic interactions on the protein surface, such as ion pairs, contribute little to protein stability, although they may play important roles in conformational specificity. We found that the tenth fibronectin type III domain of human fibronectin (FNfn10) is more stable at acidic pH than neutral pH, with an apparent midpoint of transition near pH 4. Determination of pK(a)'s for all the side chain carboxyl groups of Asp and Glu residues revealed that Asp 23 and Glu 9 have an upshifted pK(a). These residues and Asp 7 form a negatively charged patch on the surface of FNfn10, with Asp 7 centrally located between Asp 23 and Glu 9, suggesting repulsive electrostatic interactions among these residues at neutral pH. Mutant proteins, D7N and D7K, in which Asp 7 was replaced with Asn and Lys, respectively, exhibited a modest but significant increase in stability at neutral pH, compared to the wild type, and they no longer showed pH dependence of stability. The pK(a)'s of Asp 23 and Glu 9 in these mutant proteins shifted closer to their respective unperturbed values, indicating that the unfavorable electrostatic interactions have been reduced in the mutant proteins. Interestingly, the wild-type and mutant proteins were all stabilized to a similar degree by the addition of 1 M sodium chloride at both neutral and acidic pH, suggesting that the repulsive interactions between the carboxyl groups cannot be effectively shielded by 1 M sodium chloride. These results indicate that repulsive interactions between like charges on the protein surface can destabilize a protein, and protein stability can be significantly improved by relieving these interactions.     

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.     

Linkage of thioredoxin stability to titration of ionizable groups with perturbed pKa

The highly conserved, buried, Asp 26 in Escherichia coli thioredoxin has a pKa = 7.5, and its titration is associated with a sizable destabilization of the protein [Langsetmo, K., Fuchs, J., & Woodward, C. (1991) Biochemistry (preceding paper in this issue)]. A fit of the experimental pH dependence of thioredoxin stability to a theoretical expression for the pH/stability relation in proteins agrees closely with a pKa value of 7.5 for Asp 26. The agreement between the experimental and theoretical changes in protein stability due to substitution of Asp 26 by alanine is also good. The local structure in the vicinity of Asp 26 in the low-pH crystal structure (with uncharged Asp 26) is hydrophobic, indicating that the aspartate would be highly destabilized. In theoretical calculations, the desolvation penalty for deprotonating Asp 26 in this environment is similar to the total protein folding energy. As a consequence, the Asp 26 pKa would be much greater than 7.5, and/or the protein might not fold. This suggests that a compensating process partially stabilizes the Asp 26 carboxyl group when it is charged. A simple model for this proposed, whereby the Lys 57 side chain rotates to form a salt bridge with Asp 26 when it is deprotonated.