Comparison of the structures and stabilities of coiled‐coil proteins containing hexafluoroleucine and t‐butylalanine provides insight into the stabilizing effects of highly fluorinated amino acid side‐chains

Highly fluorinated analogs of hydrophobic amino acids are well known to increase the stability of proteins toward thermal unfolding and chemical denaturation, but there is very little data on the structural consequences of fluorination. We have determined the structures and folding energies of three variants of a de novo designed 4‐helix bundle protein whose hydrophobic cores contain either hexafluoroleucine (hFLeu) or t‐butylalanine (tBAla). Although the buried hydrophobic surface area is the same for all three proteins, the incorporation of tBAla causes a rearrangement of the core packing, resulting in the formation of a destabilizing hydrophobic cavity at the center of the protein. In contrast, incorporation of hFLeu, causes no changes in core packing with respect to the structure of the nonfluorinated parent protein which contains only leucine in the core. These results support the idea that fluorinated residues are especially effective at stabilizing proteins because they closely mimic the shape of the natural residues they replace while increasing buried hydrophobic surface area.     

Molecular dynamics guided study of salt bridge length dependence in both fluorinated and non-fluorinated parallel dimeric coiled-coils

The alpha-helical coiled-coil is one of the most common oligomerization motifs found in both native and engineered proteins. To better understand the stability and dynamics of the coiled-coil motifs, including those modified by fluorination, several fluorinated and nonfluorinated parallel dimeric coiled-coil protein structures were designed and modeled. We also attempt to investigate how changing the length and geometry of the important stabilizing salt bridges influences the coiled-coil protein structure. Molecular dynamics (MD) and free energy simulations with AMBER used a particle mesh Ewald treatment of the electrostatics in explicit TIP3P solvent with balanced force field treatments. Preliminary studies with legacy force fields (ff94, ff96, and ff99) show a profound instability of the coiled-coil structures in short MD simulation. Significantly, better behavior is evident with the more balanced ff99SB and ff03 protein force fields. Overall, the results suggest that the coiled-coil structures can readily accommodate the larger acidic arginine or S-2,7-diaminoheptanedoic acid mutants in the salt bridge, whereas substitution of the smaller L-ornithine residue leads to rapid disruption of the coiled-coil structure on the MD simulation time scale. This structural distortion of the secondary structure allows both the formation of large hydration pockets proximal to the charged groups and within the hydrophobic core. Moreover, the increased structural fluctuations and movement lead to a decrease in the water occupancy lifetimes in the hydration pockets. In contrast, analysis of the hydration in the stable dimeric coiled-coils shows high occupancy water sites along the backbone residues with no water occupancy in the hydrophobic core, although transitory water interactions with the salt bridge residues are evident. The simulations of the fluorinated coiled-coils suggest that in some cases fluorination electrostatically stabilizes the intermolecular coiled-coil salt bridges. Structural analyses also reveal different side chain rotamer preferences for leucine when compared with 5,5,5,5',5',5'-hexafluoroleucine mutants. These observed differences in the side chain rotamer populations suggest differential changes in the side chain conformational entropy upon coiled-coil formation when the protein is fluorinated. The free energy of hydration of the isolated 5,5,5,5',5',5'-hexafluoroleucine amino acid is calculated to be 1.1 kcal/mol less stable than leucine; this hydrophobic penalty in the monomer may provide a driving force for coiled-coil dimer formation. Estimation of the ellipticity at 222 nm from a series of snapshots from the MD simulations with DicroCalc shows distinct increases in the ellipticity when the coiled-coil is fluorinated, which suggests that the helicity in the folded coiled-coils is greater when fluorinated.     

Prediction of protein mutant stability using classification and regression tool

Prediction of protein stability upon amino acid substitutions is an important problem in molecular biology and the solving of which would help for designing stable mutants. In this work, we have analyzed the stability of protein mutants using two different datasets of 1396 and 2204 mutants obtained from ProTherm database, respectively for free energy change due to thermal (DeltaDeltaG) and denaturant denaturations (DeltaDeltaG(H(2)O)). We have used a set of 48 physical, chemical energetic and conformational properties of amino acid residues and computed the difference of amino acid properties for each mutant in both sets of data. These differences in amino acid properties have been related to protein stability (DeltaDeltaG and DeltaDeltaG(H(2)O)) and are used to train with classification and regression tool for predicting the stability of protein mutants. Further, we have tested the method with 4 fold, 5 fold and 10 fold cross validation procedures. We found that the physical properties, shape and flexibility are important determinants of protein stability. The classification of mutants based on secondary structure (helix, strand, turn and coil) and solvent accessibility (buried, partially buried, partially exposed and exposed) distinguished the stabilizing/destabilizing mutants at an average accuracy of 81% and 80%, respectively for DeltaDeltaG and DeltaDeltaG(H(2)O). The correlation between the experimental and predicted stability change is 0.61 for DeltaDeltaG and 0.44 for DeltaDeltaG(H(2)O). Further, the free energy change due to the replacement of amino acid residue has been predicted within an average error of 1.08 kcal/mol and 1.37 kcal/mol for thermal and chemical denaturation, respectively. The relative importance of secondary structure and solvent accessibility, and the influence of the dataset on prediction of protein mutant stability have been discussed.     

Packing and hydrophobicity effects on protein folding and stability: effects of beta-branched amino acids, valine and isoleucine, on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers.

The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three-dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA-binding proteins (leucine zippers), Leu residues are conserved at positions "d," and beta-branched amino acids, Ile and Val, often occur at positions "a" in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions "a" (9, 16, and 23) or "d" (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn.HCl. The results of the mutations of Ile to Val at either positions "a" or "d" in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (delta delta G values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions "a" in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (delta delta G values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic alpha-helices. These structural findings correlate with the observation that in DNA-binding proteins, the conserved Leu residues at positions "d" are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions "a."     

Four-body potentials reveal protein-specific correlations to stability changes caused by hydrophobic core mutations

Mutational experiments show how changes in the hydrophobic cores of proteins affect their stabilities. Here, we estimate these effects computationally, using four-body likelihood potentials obtained by simplicial neighborhood analysis of protein packing (SNAPP). In this procedure, the volume of a known protein structure is tiled with tetrahedra having the center of mass of one amino acid side-chain at each vertex. Log-likelihoods are computed for the 8855 possible tetrahedra with equivalent compositions from structural databases and amino acid frequencies. The sum of these four-body potentials for tetrahedra present in a given protein yields the SNAPP score. Mutations change this sum by changing the compositions of tetrahedra containing the mutated residue and their related potentials. Linear correlation coefficients between experimental mutational stability changes, Delta(DeltaG(unfold)), and those based on SNAPP scoring range from 0.70 to 0.94 for hydrophobic core mutations in five different proteins. Accurate predictions for the effects of hydrophobic core mutations can therefore be obtained by virtual mutagenesis, based on changes to the total SNAPP likelihood potential. Significantly, slopes of the relation between Delta(DeltaG(unfold)) and DeltaSNAPP for different proteins are statistically distinct, and we show that these protein-specific effects can be estimated using the average SNAPP score per residue, which is readily derived from the analysis itself. This result enhances the predictive value of statistical potentials and supports previous suggestions that "comparable" mutations in different proteins may lead to different Delta(DeltaG(unfold)) values because of differences in their flexibility and/or conformational entropy.     

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 coiled-coil peptide domains by introduction of trifluoroleucine

Substitution of leucine residues by 5,5,5-trifluoroleucine at the d-positions of the leucine zipper peptide GCN4-p1d increases the thermal stability of the coiled-coil structure. The midpoint thermal unfolding temperature of the fluorinated peptide is elevated by 13 degrees C at 30 microM peptide concentration. The modified peptide is more resistant to chaotropic denaturants, and the free energy of folding of the fluorinated peptide is 0.5-1.2 kcal/mol larger than that of the hydrogenated form. A similarly fluorinated form of the DNA-binding peptide GCN4-bZip binds to target DNA sequences with affinity and specificity identical to those of the hydrogenated form, while demonstrating enhanced thermal stability. Molecular dynamics simulation on the fluorinated GCN4-p1d peptide using the Surface Generalized Born implicit solvation model revealed that the coiled-coil binding energy is 55% more favorable upon fluorination. These results suggest that fluorination of hydrophobic substructures in peptides and proteins may provide new means of increasing protein stability, enhancing protein assembly, and strengthening receptor-ligand interactions.     

The fluorous effect in proteins: properties of alpha4F6, a 4-alpha-helix bundle protein with a fluorocarbon core

To test the prediction that extensively fluorinated (fluorous) proteins should be more stable and exhibit novel self-segregating behavior, the properties of the de novo designed model 4-alpha-helix bundle protein, alpha 4F 6, in which the hydrophobic core is packed entirely with the extensively fluorinated amino acid l-5,5,5,5',5',5'-hexafluoroleucine, have been compared with its nonfluorinated counterpart, alpha 4H, in which the core is packed with leucine. alpha 4F 6 exhibits much greater resistance to proteolysis by either chymotrypsin or trypsin than alpha 4H and resists unfolding by organic solvents far better than alpha 4H. Whereas increasing concentrations of ethanol or 2-propanol cause the helices of the alpha 4H tetramer first to dissociate into monomeric helices and then to completely unfold, these solvents have little effect on the structure of alpha 4F 6. In contrast, increasing the concentrations of the fluorinated alcohol trifluoroethanol promotes dissociation of both alpha 4H and alpha 4F 6 to monomeric helices, whereas the secondary structure of both peptides remains intact. (19)F NMR experiments indicate that the two peptides can form mixed alpha-helical alpha 4F 6:alpha 4H bundles and thus do not exhibit the self-segregating behavior predicted by the fluorous effect. We conclude that the properties of alpha 4F 6 are best explained by the more hydrophobic nature of the hexafluoroleucine side chain, rather than the low solubility of fluorocarbons in hydrocarbon solvents that forms the basis of the fluorous effect.     

Energetics of side chain packing in staphylococcal nuclease assessed by exchange of valines, isoleucines, and leucines

To examine the importance of side chain packing to protein stability, each of the 11 leucines in staphylococcal nuclease was substituted with isoleucine and valine. The nine valines were substituted with leucine and isoleucine, while the five isoleucines, previously substituted with valine, were substituted with leucine and methionine. These substitutions conserve the hydrophobic character of these side chains but alter side chain geometry and, in some cases, size. In addition, eight threonine residues, previously substituted with valine, were substituted with isoleucine to test the importance of packing at sites normally not occupied by a hydrophobic residue. The stabilities of these 58 mutant proteins were measured by guanidine hydrochloride denaturation. To the best of our knowledge, this is the largest library of single packing mutants yet characterized. As expected, repacking stability effects are tied to the degree of side chain burial. The average energetic cost of moving a single buried methyl group was 0.9 kcal/mol, albeit with a standard deviation of 0.8 kcal/mol. This average is actually slightly greater than the value of 0.7-0.8 kcal/mol estimated for the hydrophobic transfer energy of a methylene from octanol to water. These results appear to indicate that van der Waals interactions gained from optimal packing are at least as important in stabilizing the native state of proteins as hydrophobic transfer effects.     

How protein stability and new functions trade off

Numerous studies have noted that the evolution of new enzymatic specificities is accompanied by loss of the protein's thermodynamic stability (DeltaDeltaG), thus suggesting a tradeoff between the acquisition of new enzymatic functions and stability. However, since most mutations are destabilizing (DeltaDeltaG>0), one should ask how destabilizing mutations that confer new or altered enzymatic functions relative to all other mutations are. We applied DeltaDeltaG computations by FoldX to analyze the effects of 548 mutations that arose from the directed evolution of 22 different enzymes. The stability effects, location, and type of function-altering mutations were compared to DeltaDeltaG changes arising from all possible point mutations in the same enzymes. We found that mutations that modulate enzymatic functions are mostly destabilizing (average DeltaDeltaG = +0.9 kcal/mol), and are almost as destabilizing as the "average" mutation in these enzymes (+1.3 kcal/mol). Although their stability effects are not as dramatic as in key catalytic residues, mutations that modify the substrate binding pockets, and thus mediate new enzymatic specificities, place a larger stability burden than surface mutations that underline neutral, non-adaptive evolutionary changes. How are the destabilizing effects of functional mutations balanced to enable adaptation? Our analysis also indicated that many mutations that appear in directed evolution variants with no obvious role in the new function exert stabilizing effects that may compensate for the destabilizing effects of the crucial function-altering mutations. Thus, the evolution of new enzymatic activities, both in nature and in the laboratory, is dependent on the compensatory, stabilizing effect of apparently "silent" mutations in regions of the protein that are irrelevant to its function.     

Role of the minor energetic determinants of chicken egg white lysozyme (HEWL) to the stability of the HEWL.antibody scFv-10 complex

Seven of the 13 non-glycine contact amino acids in the hen (chicken) egg white lysozyme (HEWL) epitope for antibody Fab-10 each contribute < or =0.3 kcal/mol to the change in free energy (DeltaDeltaG(D)) from wild type (WT) when replaced by alanine (nullspots), and three others each give (0.7 < DeltaDeltaG(D) < or = 1. 0) kcal/mol (warm spots) (Rajpal et al. Protein Sci 1998;7:1868-1874). The low DeltaDeltaG(D) values introduced by alanine mutations present an opportunity to explore accurately their cumulative effects, as the sum of the combined DeltaDeltaG(D) values is not so large as to destabilize the complex beyond the range of accurate measurement. Substitution of six of the seven null spot residues by alanine leads to a cumulative DeltaDeltaG(D) = 2.25 +/- 0.04 kcal/mol, whereas the sum of the six individual changes is only -0.36 +/- 0.32 kcal/mol. The triple warm spot mutation generates a DeltaDeltaG(D) = 5.11 +/- 0.06 kcal/mol versus DeltaDeltaG(D) = 2.52 +/- 0.22 kcal/mol for the sum of the three individuals. The non-additivity in the individual DeltaDeltaG(D) values for the alanine mutations may indicate that these residues provide a conformationally stabilizing effect on the hot spot residues, each of which exhibits DeltaDeltaG(D) > 4.0 kcal/mol on alanine substitution.     

On the unyielding hydrophobic core of villin headpiece

Villin headpiece (HP67) is a small, autonomously-folding domain that has become a model system for understanding the fundamental tenets governing protein folding. In this communication, we explore the role that Leu61 plays in the structure and stability of the construct. Deletion of Leu61 results in a completely unfolded protein that cannot be expressed in Escherichia coli. Omission of only the aliphatic leucine side chain (HP67 L61G) perturbed neither the backbone conformation nor the orientation of local hydrophobic side chains. As a result, a large, solvent-exposed hydrophobic pocket, a negative replica of the leucine side-chain, was created on the surface. The loss of the hydrophobic interface between leucine 61 and the hydrophobic pocket destabilized the construct by ~3.3 kcal/mol. Insertion of a single glycine residue immediately before Leu61 (HP67 L61[GL]) was also highly destabilizing and had the effect of altering the backbone conformation (α-helix to π-helix) in order to precisely preserve the wild-type position and conformation of all hydrophobic residues, including Leu61. In addition to demonstrating that the hydrophobic side-chain of Leu61 is critically important for the stability of villin headpiece, our results are consistent with the notion that the precise interactions present within the hydrophobic core, rather than the hydrogen bonds that define the secondary structure, specify a protein's fold.     

Thermodynamic stability of the bacteriorhodopsin lattice as measured by lipid dilution

To determine the strength of noncovalent interactions that stabilize a membrane protein complex, we have developed an in vitro method for quantifying the dissociation of the bacteriorhodopsin (BR) lattice, a naturally occurring two-dimensional crystal. A lattice suspension was titrated with a short- and long-chain phosphatidylcholine mixture to dilute BR within the lipid bilayer. The fraction of BR in the lattice form as a function of added lipid was determined by visible circular dichroism spectroscopy and fit with a cooperative self-assembly model to obtain a critical concentration for lattice assembly. Critical concentration values of wild-type and mutant proteins were used to calculate the change in lattice stability upon mutation (DeltaDeltaG). By using this method, a series of mutant proteins was examined in which residues at the BR-BR interface were replaced with smaller amino acids, either Ala or Gly. Most of the mutant lattices were destabilized, with DeltaDeltaG values of 0.2-1.1 kcal/mol at 30 degrees C, consistent with favorable packing of apolar residues in the membrane. One mutant, I45A, was stabilized by approximately 1.0 kcal/mol, possibly due to increased lipid entropy. The DeltaDeltaG values agreed well with previous in vivo measurements, except in the case of I45A. The ability to measure the change in stability of mutant protein complexes in a lipid bilayer may provide a means of determining the contributions of specific protein-protein and protein-lipid interactions to membrane protein structure.     

Impact of proline residues on parvalbumin stability

Despite its higher net charge and reduced opportunities for favorable tertiary interactions, Ca(2+)-free rat beta-parvalbumin is more stable than rat alpha-parvalbumin. Under conditions wherein alpha denatures at 45.8 degrees C, beta denatures at 53.6 degrees. The homologous chicken beta isoform known as CPV3 also exhibits heightened stability-prompting an inquiry into the stabilizing influence of Pro-21 and Pro-26. Individual P21A and P26A mutations lower the T(m) of rat beta by 3.2 degrees, decreasing conformational stability by 0.74 kcal/mol. Simultaneous replacement of Pro-21 and Pro-26 essentially abolishes the excess stability (DeltaT(m) = -7.6 degrees; DeltaDeltaG(conf) = -1.77 kcal/mol). Significantly, the P21A/P26A variant displays Ca(2+) affinity virtually indistinguishable from wild-type beta, implying that structural alterations in the AB domain do not necessarily influence the divalent ion affinity of the CD-EF domain. The consequences of introducing proline at positions 21 and 26 in rat alpha were also examined. Whereas the H26P mutation raises the T(m) by 5.6 degrees (DeltaDeltaG(conf) = 1.25 kcal/mol), A21P lowers the T(m) by 8.5 degrees (DeltaDeltaG(conf) = -1.9 kcal/mol). Replacement of Ala-21 by proline in an alpha AB/beta CD-EF chimera increases the T(m) by 5.8 degrees (DeltaDeltaG(conf) = 0.95 kcal/mol), implying that the destabilization of alpha by Pro-21 results from steric conflict with a residue in the CD-EF domain. Consistent with that hypothesis, the K80S mutation markedly stabilizes alpha A21P, yielding a protein with a T(m) 2.0 degrees higher than wild-type alpha. The observed differences in stability resulting from proline addition/removal are largely consistent with alterations in main-chain and side-chain conformational entropy.     

Solvation and the hidden thermodynamics of a zinc finger probed by nonstandard repair of a protein crevice

The classical Zn finger contains a phenylalanine at the crux of its three architectural elements: a beta-hairpin, an alpha-helix, and a Zn(2+)-binding site. Surprisingly, phenylalanine is not required for high-affinity Zn2+ binding, but instead contributes to the specification of a precise DNA-binding surface. Substitution of phenylalanine by leucine leads to a floppy but native-like structure whose Zn affinity is maintained by marked entropy-enthalpy compensation (DeltaDeltaH -8.3 kcal/mol and -TDeltaDeltaS 7.7 kcal/mol). Phenylalanine and leucine differ in shape, size, and aromaticity. To distinguish which features correlate with dynamic stability, we have investigated a nonstandard finger containing cyclohexanylalanine at this site. The structure of the nonstandard finger is similar to that of the native domain. The cyclohexanyl ring assumes a chair conformation, and conformational fluctuations characteristic of the leucine variant are damped. Although the nonstandard finger exhibits a lower affinity for Zn2+ than does the native domain (DeltaDeltaG -1.2 kcal/mol), leucine-associated perturbations in enthalpy and entropy are almost completely attenuated (DeltaDeltaH -0.7 kcal/mol and -TDeltaDeltaS -0.5 kcal/mol). Strikingly, global changes in entropy (as inferred from calorimetry) are in each case opposite in sign from changes in configurational entropy (as inferred from NMR). This seeming paradox suggests that enthalpy-entropy compensation is dominated by solvent reorganization rather than nominal molecular properties. Together, these results demonstrate that dynamic and thermodynamic perturbations correlate with formation or repair of a solvated packing defect rather than type of physical interaction (aromatic or aliphatic) within the core.     

Probing backbone hydrogen bonds in the hydrophobic core of GCN4

Backbone amide hydrogen bonds play a central role in protein secondary and tertiary structure. Previous studies have shown that substitution of a backbone ester (-COO-) in place of a backbone amide (-CONH-) can selectively destabilize backbone hydrogen bonds in a protein while maintaining a similar conformation to the native backbone structure. The majority of these studies have focused on backbone substitutions that were accessible to solvent. The GCN4 coiled coil domain is an example of a stable alpha-helical dimer that possesses a well-packed hydrophobic core. Amino acids in the a and d positions of the GCN4 helix, which pack the hydrophobic core, were replaced with the corresponding alpha-hydroxy acids in the context of a chemoselectively ligated heterodimer. While the overall structure and oligomerization state of the heterodimer were maintained, the overall destabilization of the ester analogues was greater (average DeltaDeltaG of 3+ kcal mol(-1)) and more variable than previous studies. Since burial of the more hydrophobic ester should stabilize the backbone and reduce the DeltaDeltaG, the increased destabilization must come from another source. However, the observed destabilization is correlated with the protection factors for individual amide hydrogens from previous hydrogen exchange experiments. Therefore, our results suggest that backbone engineering through ester substitution is a useful approach for probing the relative strength of backbone hydrogen bonds.     

Thermostabilization of Escherichia coli ribonuclease HI by replacing left-handed helical Lys95 with Gly or Asn.

From the systematic replacements of amino acid residues of Escherichia coli ribonuclease HI with those of its thermophilic counterpart, the basic protrusion domain including region 6 (R6) from residues 91 to 95 was found to increase the structural stability of the mutant protein (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., and Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Further mutagenesis concentrating in the R6 region has revealed that replacements of Lys95 at the left-handed structure with Gly or Asn essentially enhances the protein stability. Gly and Asn substitutions stabilize the protein up to 1.9 kcal/mol and 0.9 kcal/mol in the free energy changes of unfolding, respectively. We propose that the amino acid substitution of left-handed non-Gly residue with Gly or Asn residue can be used as one of the general strategies to enhance protein stability, when such a non-Gly residue itself does not seriously contribute to protein stability.     

Relationship between local and global stabilities of proteins: site-directed mutants and chemically-modified derivatives of cytochrome c.

The methionine 80 sulfur-heme iron bond of rat cytochrome c, whose stability is decreased by mutating the phylogenetically invariant residue proline 30 to alanine and increased when tyrosine 67 is changed to phenylalanine, recovers its wild-type characteristics when both substitutions are performed on the same molecule. Titrations with urea, analyzed according to the heteropolymer theory [Alonso, D. O. V., & Dill, K. A. (1991) Biochemistry 30, 5974-5985], indicate that both single mutations increase the solvent exposure of hydrophobic groups in the unfolded state, while in the double mutant this conformational perturbation disappears. Similar increases in solvent exposure of hydrophobic groups are observed when the sulfur-iron bond of the wild-type protein is broken by alkylation of the methionine sulfur, by high pH, or by binding the heme iron with cyanide. The compensatory effects of the two single mutations do not extend to the overall stability of the protein. The added loss of conformational stability due to the single mutations amounts to 7.3 kcal/mol out of the 9 kcal/mol representing the overall free energy of stabilization of the native conformation of the wild-type protein. The folded conformation of the doubly mutated protein is only 2 kcal/mol less stable than that of the wild type. These results indicate that the double mutant protein is able to retain the essential folding pattern of cytochrome c and the thermodynamic stability of the methionine sulfur-heme iron bond, in spite of structural differences that weaken the overall stability of the molecule.     

Contribution of hydrophobic interactions to protein stability

Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin headpiece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compared our results with those of previous studies and reached the following conclusions: (1) Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6 ± 0.3 kcal/mol per -CH₂- group), than to the stability of a large protein, VlsE (1.6 ± 0.3 kcal/mol per -CH₂- group). (2) Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kilocalories per mole) Phe18 (3.9), Met13 (3.1), Phe7 (2.9), Phe11 (2.7), and Leu21 (2.7). (3) Based on the Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a -CH₂- group on folding contributes, on average, 1.1 ± 0.5 kcal/mol to protein stability. (4) The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔG_tr values from water to cyclohexane. (5) For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60 ± 4% and hydrogen bonds contribute 40 ± 4% to protein stability. (6) Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominantly by hydrophobic interactions.