Energetics of a hydrogen bond (charged and neutral) and of a cation-pi interaction in apoflavodoxin.

Anabaena apoflavodoxin contains a single histidine residue (H34) that interacts with two aromatic residues (F7 and Y47). The histidine and phenylalanine rings are almost coplanar and they can establish a cation-pi interaction when the histidine is protonated. The histidine and tyrosine side-chains are engaged in a hydrogen bond, which is their only contact. We analyse the energetics of these interactions using p Ka-shift analysis, double-mutant cycle analysis at two pH values, and X-ray crystallography. The H/F interaction is very weak when the histidine is neutral, but it is strengthened by 0.5 kcal mol-1on histidine protonation. Supporting this fact, the histidine p Kain a F7L mutant is 0.4 pH units lower than in wild-type. The strength of the H/Y hydrogen bond is 0.7 kcal mol-1when the histidine is charged, and it becomes stronger (1.3 kcal mol-1) when the histidine is neutral. This is consistent with our observation that the (H34)Nepsilon2-OH(Y47) distance is slightly shorter in the apoflavodoxin structure at pH 9.0 than in the previously reported structure at pH 6.0. It is also consistent with a histidine p Kavalue 0.6 pH units higher in a Y47F mutant than in the wild-type protein. We suggest that the higher stability of the neutral hydrogen bond could be due to a higher desolvation penalty of the charged hydrogen bond that would offset its more favourable enthalpy of formation. The relationship between hydrogen bond strength and the contribution of hydrogen bonds to protein stability is discussed.     

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.     

Contribution of hydrogen bonds to the conformational stability of human lysozyme: calorimetry and X-ray analysis of six Ser --> Ala mutants.

To further examine the contribution of hydrogen bonds to the conformational stability of the human lysozyme, six Ser to Ala mutants were constructed. The thermodynamic parameters for denaturation of these six Ser mutant proteins were investigated by differential scanning calorimetry (DSC), and the crystal structures were determined by X-ray analysis. The denaturation Gibbs energy (DeltaG) of the Ser mutant proteins was changed from 2.0 to -5.7 kJ/mol, compared to that of the wild-type protein. With an analysis in which some factors that affected the stability due to mutation were considered, the contribution of hydrogen bonds to the stability (Delta DeltaGHB) was extracted on the basis of the structures of the mutant proteins. The results showed that hydrogen bonds between protein atoms and between a protein atom and a water bound with the protein molecule favorably contribute to the protein stability. The net contribution of one intramolecular hydrogen bond to protein stability (DeltaGHB) was 8.9 +/- 2.6 kJ/mol on average. However, the contribution to the protein stability of hydrogen bonds between a protein atom and a bound water molecule was smaller than that for a bond between protein atoms.     

An energetic scale for equilibrium H/D fractionation factors illuminates hydrogen bond free energies in proteins

Equilibrium H/D fractionation factors have been extensively employed to qualitatively assess hydrogen bond strengths in protein structure, enzyme active sites, and DNA. It remains unclear how fractionation factors correlate with hydrogen bond free energies, however. Here we develop an empirical relationship between fractionation factors and free energy, allowing for the simple and quantitative measurement of hydrogen bond free energies. Applying our empirical relationship to prior fractionation factor studies in proteins, we find: [1] Within the folded state, backbone hydrogen bonds are only marginally stronger on average in α‐helices compared to β‐sheets by ～0.2 kcal/mol. [2] Charge‐stabilized hydrogen bonds are stronger than neutral hydrogen bonds by ～2 kcal/mol on average, and can be as strong as –7 kcal/mol. [3] Changes in a few hydrogen bonds during an enzyme catalytic cycle can stabilize an intermediate state by –4.2 kcal/mol. [4] Backbone hydrogen bonds can make a large overall contribution to the energetics of conformational changes, possibly playing an important role in directing conformational changes. [5] Backbone hydrogen bonding becomes more uniform overall upon ligand binding, which may facilitate participation of the entire protein structure in events at the active site. Our energetic scale provides a simple method for further exploration of hydrogen bond free energies.     

Contribution of intra- and intermolecular hydrogen bonds to the conformational stability of human lysozyme(,).

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.     

Native-specific stabilization of flavodoxin by the FMN cofactor: structural and thermodynamical explanation

Flavodoxins are useful models to investigate protein/cofactor interactions. The binding energy of the apoflavodoxin-FMN complex is high and therefore the holoflavodoxin is expected to be more stable than the apoprotein. This expectation has been challenged by reports on the stability of Desulfovibrio desulfuricans flavodoxin indicating that FMN binds to the unfolded polypeptide with similar affinity as to the native state, thus causing no net effect on protein stability. In previous work, we have analyzed in detail the stability of the apoflavodoxin from Anabaena PCC 7119 and the energetics of its functional complex with FMN. Here, we use the Anabaena holoprotein to directly investigate the contribution of the bound cofactor to protein stability through a detailed analysis of the chemical and thermal denaturation equilibria. Our data clearly shows that FMN binding largely stabilizes the protein towards both chemical and thermal denaturation, and that the stabilization observed at 25 degrees C in low ionic strength conditions is precisely the one expected if full release of the cofactor takes place upon flavodoxin unfolding. On the other hand, the binding of FMN to the native polypeptide is shown to simplify the thermal unfolding so that, while apoflavodoxin follows a three-state mechanism, the holoprotein unfolds in a two-state fashion. Comparison of the X-ray structure of native apoflavodoxin with the phi-structure of the thermal intermediate indicates that the increase in cooperativity driven by the cofactor originates in its preferential binding to the native state, which is a consequence of the disorganization in the intermediate of the FMN binding loops and of an adjacent longer loop.     

Stabilization of apoflavodoxin by replacing hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or Gln

Knowledge of protein stability principles provides a means to increase protein stability in a rational way. Here we explore the feasibility of stabilizing proteins by replacing solvent-exposed hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or GLN: The rationale behind this is a previous observation that, in some cases, neutral hydrogen bonds may be more stable that charged ones. We identified, in the apoflavodoxin from Anabaena PCC 7119, three surface-exposed aspartate or glutamate residues involved in hydrogen bonding with a single partner and we mutated them to asparagine or glutamine, respectively. The effect of the mutations on apoflavodoxin stability was measured by both urea and temperature denaturation. We observed that the three mutant proteins are more stable than wild-type (on average 0.43 kcal/mol from urea denaturation and 2.8 degrees C from a two-state analysis of fluorescence thermal unfolding data). At high ionic strength, where potential electrostatic repulsions in the acidic apoflavodoxin should be masked, the three mutants are similarly more stable (on average 0.46 kcal/mol). To rule out further that the stabilization observed is due to removal of electrostatic repulsions in apoflavodoxin upon mutation, we analysed three control mutants and showed that, when the charged residue mutated to a neutral one is not hydrogen bonded, there is no general stabilizing effect. Replacing hydrogen-bonded charged Asp or Glu residues by Asn or Gln, respectively, could be a straightforward strategy to increase protein stability.     

Effect of deleterious nsSNP on the HER2 receptor based on stability and binding affinity with herceptin: a computational approach

In this study, we identified the most deleterious non-synonymous SNP of ERBB2 (HER2) receptors by its stability and investigated its binding affinity with herceptin. Out of 135 SNPs, 10 are nsSNPs in the coding region, in which one of the nsSNP (SNPid rs4252633) is commonly found to be damaged by I-Mutant 2.0, SIFT and PolyPhen servers. With this effort, we modelled the mutant HER2 protein based on this deleterious nsSNP (rs4252633). The modeled mutant showed less stability than native HER 2 protein, based on both total energy of the mutant and stabilizing residues in the mutant protein. This is due to a deviation between the mutant and the native HER2, having an RMSD of about 2.81 A. Furthermore, we compared the binding efficiency of herceptin with native and mutant HER2 receptors. We found that herceptin has a high binding affinity with mutant HER2 receptor, with a binding energy of -24.40 kcal/mol, as compared to the native type, which has a binding energy of -15.26 kcal/mol due to six-hydrogen bonding and two salt bridges exist between herceptin and the mutant type, whereas the native type establishes four hydrogen bonds and two salt bridges with herceptin. This analysis portrays that mutant type has two additional hydrogen bonds with herceptin compared with the native type. Normal mode analysis also showed that the two amino acids, namely Asp596 and Glu598 of mutant HER2, forming additional hydrogen bonding with herceptin, had a slightly higher flexibility than the native type. Based on our investigations, we propose that SNPid rs4252633 could be the most deleterious nsSNP for HER2 receptor, and that herceptin could be the best drug for mutant compared to the native HER2 target.     

Contribution of polar groups in the interior of a protein to the conformational stability

It has been generally believed that polar residues are usually located on the surface of protein structures. However, there are many polar groups in the interior of the structures in reality. To evaluate the contribution of such buried polar groups to the conformational stability of a protein, nonpolar to polar mutations (L8T, A9S, A32S, I56T, I59T, I59S, A92S, V93T, A96S, V99T, and V100T) in the interior of a human lysozyme were examined. The thermodynamic parameters for denaturation were determined using a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. If a polar group had a heavy energy cost to be buried, a mutant protein would be remarkably destabilized. However, the stability (Delta G) of the Ala to Ser and Val to Thr mutant human lysozymes was comparable to that of the wild-type protein, suggesting a low-energy penalty of buried polar groups. The structural analysis showed that all polar side chains introduced in the mutant proteins were able to find their hydrogen bond partners, which are ubiquitous in protein structures. The empirical structure-based calculation of stability change (Delta Delta G) [Takano et al. (1999) Biochemistry 38, 12698--12708] revealed that the mutant proteins decreased the hydrophobic effect contributing to the stability (Delta G(HP)), but this destabilization was recovered by the hydrogen bonds newly introduced. The present study shows the favorable contribution of polar groups with hydrogen bonds in the interior of protein molecules to the conformational stability.     

Increasing Protein Stability by Polar Surface Residues: Domain-Wide Consequences of Interactions Within a Loop

We have examined the influence of surface hydrogen bonds on the stability of proteins by studying the effects of mutations of human immunoglobulin light chain variable domain (V_L). In addition to the variants Y27dD, N28F, and T94H of protein κIV Len that were previously described, we characterized mutants M4L, L27cN, L27cQ, and K39T, double mutant M4L/Y27dD, and triple mutant M4L/Y27dD/T94H. The triple mutant had an enhanced thermodynamic stability of 4.2 kcal/mol. We determined the structure of the triple mutant by x-ray diffraction and correlated the changes in stability due to the mutations with changes in the three-dimensional structure. Y27dD mutant had increased stability of Len by 2.7 kcal/mol, a large value for a single mutation. Asp27d present in CDR1 formed hydrogen bonds with the side-chain and main-chain atoms within the loop. In the case of the K39T mutant, which reduces stability by 2 kcal/mol, Lys39 in addition to forming a hydrogen bond with a carbonyl oxygen of a neighboring loop may also favorably influence the surface electrostatics of the molecule. We showed that hydrogen bonds between residues in surface loops can add to the overall stability of the V_L domains. The contribution to stability is further increased if the surface residue makes more than one hydrogen bond or if it forms a hydrogen bond between neighboring turns or loops separated from each other in the amino acid sequence. Based on our experiments we suggest that stabilization of proteins might be systematically accomplished by introducing additional hydrogen bonds on the surface. These substitutions are more straightforward to predict than core-packing interactions and can be selected to avoid affecting the protein’s function.     

Mapping the lifetimes of local opening events in a native state protein.

The rate constants for the processes that lead to local opening and closing of the structures around hydrogen bonds in native proteins have been determined for most of the secondary structure hydrogen bonds in the four-helix protein acyl coenzyme A binding protein. In an analysis that combines these results with the energies of activation of the opening processes and the stability of the local structures, three groups of residues in the protein structure have been identified. In one group, the structures around the hydrogen bonds have frequent openings, every 600 to 1,500 s, and long lifetimes in the open state, around 1 s. In another group of local structures, the local opening is a very rare event that takes place only every 15 to 60 h. For these the lifetime in the open state is also around 1 s. The majority of local structures have lifetimes between 2,000 and 20,000 s and relatively short lifetimes of the open state in the range between 30 and 400 ms. Mapping of these groups of amides to the tertiary structure shows that the openings of the local structures are not cooperative at native conditions, and they rarely if ever lead to global unfolding. The results suggest a mechanism of hydrogen exchange by progressive local openings.     

Native hydrogen bonds in a molten globule: the apoflavodoxin thermal intermediate

The structure and energetics of protein-folding intermediates are poorly understood. We have identified, in the thermal unfolding of the apoflavodoxin from Anabaena PCC 7119, an equilibrium intermediate with spectroscopic properties of a molten globule and substantial enthalpy and heat capacity of unfolding. The structure of the intermediate is probed by mutagenesis (and phi analysis) of polar residues involved in surface-exposed hydrogen bonds connecting secondary-structure elements in the native protein. All hydrogen bonds analysed are formed in the molten globule intermediate, either with native strength or debilitated. This suggests the overall intermediate's topology and surface tertiary interactions are close to native, and indicates that hydrogen bonding may contribute significantly to shape the conformation and energetics of folding intermediates.     

Evaluation of cooperative interactions between substructures of iso-1-cytochrome c using double mutant cycles

A double mutant cycle has been used to evaluate interaction energies between the global stabilizer mutation asparagine 52 --> isoleucine (N52I) in iso-1-cytochrome c and mutations producing single surface histidines at positions 26, 33, 39, 54, 73, 89, and 100. These histidine mutation sites are distributed through the four cooperative folding units of cytochrome c. The double mutant cycle starts with the iso-1-cytochrome c variant AcTM, a variant with no surface histidines and with asparagine at position 52. Isoleucine is added singly at position 52, AcTMI52 variant, as are the surface histidines, AcHX variants, where X indicates the histidine sequence position. The double mutant variants, AcHXI52, provide the remaining corner of the double mutant cycle. The stabilities of all variants were determined by guanidine hydrochloride denaturation and interaction energies were calculated between position 52 and each histidine site. Six of the seven double mutants show additive (AcH33I52, AcH39I52, AcH54I52, AcH89I52, and AcH100I52) stability effects or weak interaction energies (AcH73I52) of the histidine mutations and the N52I mutation, consistent with cooperative effects on protein folding and stability being sparsely distributed through the protein structure. The AcH26I52 variant shows a strong favorable interaction energy, 2.0 +/- 0.5 kcal/mol, between the N52I mutation in one substructure and the addition of His 26 to an adjacent substructure. The data are consistent with an entropic stabilization of the intersubstructure hydrogen bond between His 26 and Glu 44 by the Ile 52 mutation.     

Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity.

The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels.     

Last in, first out: the role of cofactor binding in flavodoxin folding

Although many proteins require the binding of a ligand to be functional, the role of ligand binding during folding is scarcely investigated. Here, we have reported the influence of the flavin mononucleotide (FMN) cofactor on the global stability and folding kinetics of Azotobacter vinelandii holoflavodoxin. Earlier studies have revealed that A. vinelandii apoflavodoxin kinetically folds according to the four-state mechanism: I(1) <=> unfolded apoflavodoxin <=> I(2) <=> native apoflavodoxin. I(1)an off-pathway molten globule-like is intermediate that populates during denaturant-induced equilibrium unfolding; I(2) is a high energy on-pathway folding intermediate that never populates to a significant extent. Here, we have presented extensive denaturant-induced equilibrium unfolding data of holoflavodoxin, holoflavodoxin with excess FMN, and apoflavodoxin as well as kinetic folding and unfolding data of holoflavodoxin. All folding data are excellently described by a five-state mechanism: I(1) + FMN <=> unfolded apoflavodoxin + FMN <=> I(2) + FMN <=> native apoflavodoxin + FMN<=> holoflavodoxin. The last step in flavodoxin folding is thus the binding of FMN to native apoflavodoxin. I(1),I(2), and unfolded apoflavodoxin do not interact to a significantextent with FMN. The autonomous formation of native apoflavodoxin is essential during holoflavodoxin folding. Excess FMN does not accelerate holoflavodoxin folding, and FMN does not act as a nucleation site for folding. The stability of holoflavodoxin is so high that even under strongly denaturing conditions FMN needs to be released first before global unfolding of the protein can occur.     

Dissecting the roles of individual interactions in protein stability: lessons from a circularized protein

A circular form of bovine pancreatic trypsin inhibitor (BPTI) has been prepared by introducing a peptide bond between the N- and C-termini, which are in close proximity in the native conformation. The pathway and energetics of the disulphide-coupled folding transition of the circular protein have been studied using methods applied previously to the unmodified protein. The cross-link between the termini was found not to significantly stabilize the native state in spite of the expected reduction in entropy of the unfolded protein. This unexpected result has led to a reexamination of the stabilization expected from a cross-link, considering effects on the native, as well as unfolded, states of the protein. The greatest stabilization is expected when the cross-linked groups are held rigidly in the native protein in the optimum orientation for forming the cross-link. Similar analyses, utilizing thermodynamic cycles, can be applied to other interactions that stabilize native proteins, including disulphide bonds, salt bridges, and hydrogen bonds and to modifications to the protein that remove them. In general, the contribution of an individual interaction to the stability of the native state depends on the extent to which the interaction is favored in the native conformation, which can vary greatly depending on the local environment of the interacting groups.     

Cooperative hydrogen bond interactions in the streptavidin-biotin system

The thermodynamic and structural cooperativity between the Ser45- and D128-biotin hydrogen bonds was measured by calorimetric and X-ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity approximately 2x10(7) times lower than that of wild-type streptavidin at 25 degrees C. The corresponding reduction in binding free energy (DeltaDeltaG) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11-fold greater than that predicted by a linear combination of the single-mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128-biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild-type hydrogen bond between D128-OD and biotin-N2 is thermodynamically stronger than that between S45-OG and biotin-N1.     

Multiple binding modes for palmitate to barley lipid transfer protein facilitated by the presence of proline 12

Molecular dynamics simulations have been used to characterise the binding of the fatty acid ligand palmitate in the barley lipid transfer protein 1 (LTP) internal cavity. Two different palmitate binding modes (1 and 2), with similar protein–ligand interaction energies, have been identified using a variety of simulation strategies. These strategies include applying experimental protein–ligand atom–atom distance restraints during the simulation, or protonating the palmitate ligand, or using the vacuum GROMOS 54B7 force‐field parameter set for the ligand during the initial stages of the simulations. In both the binding modes identified the palmitate carboxylate head group hydrogen bonds with main chain amide groups in helix A, residues 4 to 19, of the protein. In binding mode 1 the hydrogen bonds are to Lys 11, Cys 13, and Leu 14 and in binding mode 2 to Thr 15, Tyr 16, Val 17, Ser 24 and also to the OH of Thr 15. In both cases palmitate binding exploits irregularity of the intrahelical hydrogen‐bonding pattern in helix A of barley LTP due to the presence of Pro 12. Simulations of two variants of barley LTP, namely the single mutant Pro12Val and the double mutant Pro12Val Pro70Val, show that Pro 12 is required for persistent palmitate binding in the LTP cavity. Overall, the work identifies key MD simulation approaches for characterizing the details of protein–ligand interactions in complexes where NMR data provide insufficient restraints.     

Hydrogen bonding increases packing density in the protein interior

The contribution of hydrogen bonds and the burial of polar groups to protein stability is a controversial subject. Theoretical studies suggest that burying polar groups in the protein interior makes an unfavorable contribution to the stability, but experimental studies show that burying polar groups, especially those that are hydrogen bonded, contributes favorably to protein stability. Understanding the factors that are not properly accounted for by the theoretical models would improve the models so that they more accurately describe experimental results. It has been suggested that hydrogen bonds may contribute to protein stability, in part, by increasing packing density in the protein interior, and thereby increasing the contribution of van der Waals interactions to protein stability. To investigate the influence of hydrogen bonds on packing density, we analyzed 687 crystal structures and determined the volume of buried polar groups as a function of their extent of hydrogen bonding. Our findings show that peptide groups and polar side chains that form hydrogen bonds occupy a smaller volume than the same groups when they do not form hydrogen bonds. For example, peptide groups in which both polar groups are hydrogen bonded occupy a volume, on average, 5.2 A3 less than a peptide group that is not hydrogen bonded.     

Correlations between kinetic and X-ray analyses of engineered enzymes: crystal structures of mutants Cys----Gly-35 and Tyr----Phe-34 of tyrosyl-tRNA synthetase

The crystal structures of two mutant tyrosyl-tRNA synthetases (TyrTS) are reported to test predictions from kinetic data about structural perturbations and also to aid in the interpretation of apparent strengths of hydrogen bonds measured by protein engineering. The enzyme-tyrosine and enzyme-tyrosyl adenylate complexes of the mutant, TyrTS(Cys----Gly-35), have been determined at 2.5- and 2.7-A resolution, respectively. Residue Cys-35 is in the ribose binding site. Small rearrangements in structure are seen in the enzyme-tyrosine complex that are localized around the cavity created by the mutation. The side chain of Thr-51 moves to occupy the cavity, and Ile-52 adopts two significantly populated conformations, one as in the native enzyme and a second unique to the mutant. On binding tyrosyl adenylate, Ile-52 in the mutant crystal structure preferentially occupies the conformation observed in the native structure. The side chain at Thr-51 becomes disordered. The double-mutant test, which was designed to detect interactions between residues, had previously shown a discrepancy of some 0.4 kcal/mol on mutating Cys-35 and Thr-51 separately and together. A crystal structure of a second mutant, delta TyrTS(Tyr----Phe-34), complexed with tyrosine has been determined at 2.7-A resolution. Tyr-34 in wild-type enzyme makes a hydrogen bond with the phenolic oxygen of the bound tyrosine substrate. The mutant crystal structure was solved to discover whether or not a water molecule binds to the substrate instead of the hydroxyl of Tyr-34 as the interpretation of apparent binding energies from site-directed mutagenesis experiments hinges crucially on whether there is access of water to the mutated region.