Fine structure analysis of a protein folding transition state; distinguishing between hydrophobic stabilization and specific packing

Developing a detailed understanding of the structure and energetics of protein folding transition states is a key step in describing the folding process. The phi-value analysis approach allows the energetic contribution of side-chains to be mapped out by comparing wild-type with individual mutants where conservative changes are introduced. Studies where multiple substitutions are made at individual sites are much rarer but are potentially very useful for understanding the contribution of each element of a side-chain to transition state formation, and for distinguishing the relative importance of specific packing versus hydrophobic interactions. We have made a series of conservative mutations at multiple buried sites in the N-terminal domain of L9 in order to assess the relative importance of specific side-chain packing versus less specific hydrophobic stabilization of the transition state. A total of 28 variants were prepared using both naturally occurring and non-naturally occurring amino acids at six sites. Analysis of the mutants by NMR and CD showed no perturbation of the structure. There is no correlation between changes in hydrophobicity and changes in stability. In contrast, there is excellent linear correlation between the hydrophobicity of a side-chain and the log of the folding rate, ln(k(f)). The correlation between ln(k(f)) and the change in hydrophobicity holds even for substitutions that change the shape and/or size of a side-chain significantly. For most sites, the correlation with the logarithm of the unfolding rate, ln(k(u)), is much worse. Mutants with more hydrophobic amino acid substitutions fold faster, and those with less hydrophobic amino acid substitutions fold slower. The results show that hydrophobic interactions amongst core residues are an important driving force for forming the transition state, and are more important than specific tight packing interactions. Finally, a number of substitutions lead to negative phi-values and the origin of these effects are described.     

Pressure versus heat-induced unfolding of ribonuclease A: the case of hydrophobic interactions within a chain-folding initiation site

To investigate the characteristics of the postulated carboxy terminal chain-folding initiation site in bovine pancreatic ribonuclease A (RNase A) (residues 106-118), important in the early stages of the folding pathway, we have engineered by site-directed mutagenesis a set of 14 predominantly conservative hydrophobic variants of the protein. The stability of each variant has been compared by pressure and temperature-induced unfolding, monitored by fourth derivative UV absorbance spectroscopy. Apparently simple two-state, reversible unfolding transitions are observed, suggesting that the disruption of tertiary structure of each protein at high pressure or temperature is strongly cooperative. Within the limits of the technique, we are unable to detect significant differences between the two processes of denaturation. Both steady-state kinetic parameters for the enzyme reaction and UV CD spectra of each RNase A variant indicate that truncation of hydrophobic side chains in this region has, in general, little or no effect on the native structure and function of the enzyme. Furthermore, the decreases in free energy of unfolding upon pressure and thermal denaturation of all the variants, particularly those modified at residues 106 and 108, suggest that the hydrophobic residues and side chain packing interactions of this region play an important role in maintaining the conformational stability of RNase A. We also demonstrate the potential of Tyr115 replacement by Trp as a non-destabilizing fluorescence probe of conformational changes local to the region.     

The relationship between conservation, thermodynamic stability, and function in the SH3 domain hydrophobic core

To investigate the relationships between sequence conservation, protein stability, and protein function, we have measured the thermodynamic stability, folding kinetics, and in vitro peptide-binding activity of a large number of single-site substitutions in the hydrophobic core of the Fyn SH3 domain. Comparison of these data to that derived from an analysis of a large alignment of SH3 domain sequences revealed a very good correlation between the distinct pattern of conservation observed at each core position and the thermodynamic stability of mutants. Conservation was also found to correlate well with the unfolding rates of mutants, but not to the folding rates, suggesting that evolution selects more strongly for optimal native state packing interactions than for maximal folding rates. Structural analysis suggests that residue-residue core packing interactions are very similar in all SH3 domains, which provides an explanation for the correlation between conservation and mutant stability effects studied in a single SH3 domain. We also demonstrate a correlation between stability and the in vivo activity of mutants, and between conservation and activity. However, the relationship between conservation and activity was very strong only for the three most conserved hydrophobic core positions. The weaker correlation between activity and conservation seen at the other seven core positions indicates that maintenance of protein stability is the dominant selective pressure at these positions. In general, the pattern of conservation at hydrophobic core positions appears to arise from conserved packing constraints, and can be effectively utilized to predict the destabilizing effects of amino acid substitutions.     

Contribution of the 30/36 hydrophobic contact at the C-terminus of the alpha-helix to the stability of the ubiquitin molecule

The contribution of the hydrophobic contact in the C-capping motif of the alpha-helix to the thermodynamic stability of the ubiquitin molecule has been analyzed. For this, 16 variants of ubiquitin containing the full combinatorial set of four nonpolar residues Val, Ile, Leu, and Phe at C4 (Ile30) and C' ' (Ile36) positions were generated. The secondary structure content as estimated using far-UV circular dichroism (CD) spectroscopy of all but Phe variants at position 30 did not show notable changes upon substitutions. The thermodynamic stability of these ubiquitin variants was measured using differential scanning calorimetry, and it was shown that all variants have lower stability as measured by decreases in the Gibbs energy. Since in some cases the decrease in stability was so dramatic that it rendered an unfolded protein, it was therefore concluded that, despite apparent preservation of the secondary structure, the 30/36 hydrophobic contact is essential for the stability of the ubiquitin molecule. The decrease in the Gibbs energy in many cases was found to be accompanied by a large (up to 25%) decrease in the enthalpy of unfolding, particularly significant in the variants containing Ile to Leu substitutions. This decrease in enthalpy of unfolding is proposed to be primarily the result of the perturbed packing interactions in the native state of the Ile --> Leu variants. The analysis of these data and comparison with effects of similar amino acid substitutions on the stability of other model systems suggest that Ile --> Leu substitutions cannot be isoenergetic at the buried site.     

Thermodynamic and structural studies of cavity formation in proteins suggest that loss of packing interactions rather than the hydrophobic effect dominates the observed energetics

The hydrophobic effect is widely believed to be an important determinant of protein stability. However, it is difficult to obtain unambiguous experimental estimates of the contribution of the hydrophobic driving force to the overall free energy of folding. Thermodynamic and structural studies of large to small substitutions in proteins are the most direct method of measuring this contribution. We have substituted the buried residue Phe8 in RNase S with alanine, methionine, and norleucine. Binding thermodynamics and structures were characterized by titration calorimetry and crystallography, respectively. The crystal structures of the RNase S F8A, F8M, and F8Nle mutants indicate that the protein tolerates the changes without any main chain adjustments. The correlation of structural and thermodynamic parameters associated with large to small substitutions was analyzed for nine mutants of RNase S as well as 32 additional cavity-containing mutants of T4 lysozyme, human lysozyme, and barnase. Such substitutions were typically found to result in negligible changes in DeltaC(p)() and positive values of both DeltaDeltaH degrees and DeltaDeltaS of folding. Enthalpic effects were dominant, and the sign of DeltaDeltaS is the opposite of that expected from the hydrophobic effect. Values of DeltaDeltaG degrees and DeltaDeltaH degrees correlated better with changes in packing parameters such as residue depth or occluded surface than with the change in accessible surface area upon folding. These results suggest that the loss of packing interactions rather than the hydrophobic effect is a dominant contributor to the observed energetics for large to small substitutions. Hence, estimates of the magnitude of the hydrophobic driving force derived from earlier mutational studies are likely to be significantly in excess of the actual value.     

Comparison of heat- and pressure-induced unfolding of ribonuclease a: the critical role of Phe46 which appears to belong to a new hydrophobic chain-folding initiation site

To clarify the structural role of Phe46 inside the hydrophobic core of bovine pancreatic ribonuclease A (RNase A), thermal and pressure unfolding of wild-type RNase A and three mutant forms (F46V, F46E, and F46K) were analyzed by fourth-derivative UV absorbance spectroscopy. All the mutants, as well as the wild type, exhibited a two-state transition during both thermal and pressure unfolding, and both T(m) and P(m) decreased markedly when Phe46 was replaced with valine, glutamic acid, or lysine. The strongest effect was on the F46K mutant and the weakest on F46V. Both unfolding processes produced identical blue shifts in the fourth-derivative spectra, indicating that the tyrosine residues are similarly exposed in the temperature- and pressure-induced unfolded states. A comparison of Gibbs free energies determined from the pressure and temperature unfoldings, however, gave DeltaG(p)/DeltaG(t) ratios (r) of 1.7 for the wild type and 0.92 +/- 0.03 for the mutants. Furthermore, the DeltaV value for each mutant was larger than that for the wild type. CD spectra and activity measurements showed no obvious major structural differences in the folded state, indicating that the structures of the Phe46 mutants and wild type differ in the unfolded state. We propose a model in which Phe46 stabilizes the hydrophobic core at the boundary between two structural domains. Mutation of Phe46 decreases protein stability by weakening the unfolding cooperativity between these domains. This essential function of Phe46 in RNase A stability indicates that it belongs to a chain-folding initiation site.     

Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A

This research was undertaken to distinguish between local and global unfolding in the reversible thermal denaturation of bovine pancreatic ribonclease A (RNase A). Local unfolding was monitored by steady-state and time-resolved fluorescence of nine mutants in each of which a single tryptophan was substituted for a wild-type residue. Global unfolding was monitored by far-UV circular dichroism and UV absorbance. All the mutants (except F8W and D38W) exhibited high specific enzymatic activity, and their far-UV CD spectra were very close to that of wild-type RNase A, indicating that the tryptophan substitutions did not affect the structure of any of the mutants (excluding K1W and Y92W) under folding conditions at 20 degrees C. Like wild-type RNase A, the various mutants exhibited reversible cooperative thermal unfolding transitions at pH 5, with transition temperatures 2.5-11 degrees C lower than that of the wild-type transition, as detected by far-UV CD or UV absorbance. Even at 80 degrees C, well above the cooperative transition of all the RNase A mutants, a considerable amount of secondary and tertiary structure was maintained. These studies suggest the following two-stage mechanism for the thermal unfolding transition of RNase A as the temperature is increased. First, at temperatures lower than those of the main cooperative transition, long-range interactions within the major hydrophobic core are weakened, e.g., those involving residues Phe-8 (in the N-terminal helix) and Lys-104 and Tyr-115 (in the C-terminal beta-hairpin motif). The structure of the chain-reversal loop (residues 91-95) relaxes in the same temperature range. Second, the subsequent higher-temperature cooperative unfolding transition is associated with a loss of secondary structure and additional changes in the tertiary contacts of the major hydrophobic core, e.g., those involving residues Tyr-73, Tyr-76, and Asp-38 on the other side of the molecule. The hydrophobic interactions of the C-terminal loop of the protein are enhanced by high temperature, and perhaps are responsible for the preservation of the local structural environment of Trp-124 at temperatures slightly above the major cooperative transition. The results shed new light on the thermal unfolding transitions, generally supporting the thermal unfolding hypothesis of Burgess and Scheraga, as modified by Matheson and Scheraga.     

De novo design of the hydrophobic core of ubiquitin.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.     

Core side-chain packing and backbone conformation in Lpp-56 coiled-coil mutants

Native proteins exhibit precise geometric packing of atoms in their hydrophobic interiors. Nonetheless, controversy remains about the role of core side-chain packing in specifying and stabilizing the folded structures of proteins. Here we investigate the role of core packing in determining the conformation and stability of the Lpp-56 trimerization domain. The X-ray crystal structures of Lpp-56 mutants with alanine substitutions at two and four interior core positions reveal trimeric coiled coils in which the twist of individual helices and the helix-helix spacing vary significantly to achieve the most favored superhelical packing arrangement. Introduction of each alanine "layer" into the hydrophobic core destabilizes the superhelix by 1.4 kcal mol(-1). Although the methyl groups of the alanine residues pack at their optimum van der Waals contacts in the coiled-coil trimer, they provide a smaller component of hydrophobic interactions than bulky hydrophobic side-chains to the thermodynamic stability. Thus, specific side-chain packing in the hydrophobic core of coiled coils are important determinants of protein main-chain conformation and stability.     

Structural and thermodynamic analysis of compensating mutations within the core of chicken egg white lysozyme.

High resolution crystal structures have been determined for six chicken-type lysozymes that were constructed to investigate putative intermediates in the evolution of the lysozymes of modern game birds (Malcolm, B. A., Wilson, K. P., Matthews, B. W., Kirsch, J. F., and Wilson, A. C. (1990) Nature 345, 86-89). The amino acid replacements include Thr-40----Ser, Ile-55----Val, and Ser-91----Thr, as well as combinations of these substitutions. Residues 40, 55, and 91 are buried within the core of chicken lysozyme. The replacements therefore involve the insertion and/or removal of methyl groups from the protein interior. The mutant proteins have normal activities, and their thermal stabilities span a range of 7 degrees C, with some variants more stable and some less stable than the naturally occurring forms. Comparison of the crystal structures shows the overall structures to be very similar, but there are differences in the packing of side chains in the region of the replacements. The x-ray coordinates were used to evaluate the repacking of side chains in the protein interior and to attempt to evaluate the contributions of the different energetic interactions toward the overall stability of each variant. The results illustrate how proteins can compensate for potentially destabilizing substitutions in different ways and underscore the importance of high resolution structural data if changes in protein thermostability due to changes in protein sequence are to be understood. The findings also suggest that protein stability can be increased by mutations that lower strain in the protein interior while maintaining total buried hydrophobic surface area.     

Minimization of cavity size ensures protein stability and folding: structures of Phe46-replaced bovine pancreatic RNase A

The Phe46 residue, located in the hydrophobic core of RNase A, was replaced with other hydrophobic residues, leucine, valine, or alanine, and their X-ray crystallographic structures were determined up to 1.50-1.80 A resolution in an attempt to examine the relationship between structural changes and conformational stability or folding kinetics. The backbone structure of F46L, F46V, and F46A was indistinguishable from that of the wild-type enzyme, retaining the correct active site structure. However, one water molecule was included in the hydrophobic core of F46A, forming two hydrogen bonds with the backbone peptide chain. The side chain of Met29 in F46V and F46A adopted two different conformations in an equal occupancy. A trapped water molecule and two conformations of Met29 represent changes that minimize the cavity volume. Nevertheless, the replacement of Phe46 with the above residues resulted in a marked decrease in both thermal stability and folding reaction. Thus, Phe46 ensures the thermal stability and the rapid and correct folding of RNase A by the role it plays in forming a highly packed, hydrophobic core.     

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.     

Effects of charge-to-alanine substitutions on the stability of ribosomal protein L30e from Thermococcus celer

The ability to rationally engineer a protein with altered stability depends upon the detailed understanding of the role of noncovalent interactions in defining thermodynamic properties of proteins. In this paper, we used T. celer L30e as a model to address the question of the role of charge-charge interactions in defining the stability of this protein. A total of 26 single-site charge-to-alanine variants of this protein were generated, and the stability of these proteins was determined using thermal- and denaturant-induced unfolding. It was found that, although L30e is isolated from a thermophilic organism and is highly thermostable, some of the substitutions lead to a further increase in the transition temperature. Analysis of the effects of high ionic strength on the stabilities of L30e variants shows that the long-range charge-charge interactions are as important as the short-range (salt bridge) interactions. The changes in stabilities of the T. celer L30e protein variants were compared with the changes in the energy of charge-charge interactions calculated using different computational models. It was found that there is a good qualitative agreement between experimental and calculated data: for 70-80% (19-21 of 26, confidence p < 0.003) of the variants, computational models predict correctly the sign of the stability changes. In particular, computational models identify correctly those charged amino acid residue substitutions of which led to enhancement in thermostability. Thus, optimization of the charge-charge interactions might be a useful approach for the rational increase in protein stability.     

Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis alpha-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a beta-sheet by introducing a residue with high beta-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.     

A thermodynamic and kinetic analysis of the folding pathway of an SH3 domain entropically stabilised by a redesigned hydrophobic core

The folding thermodynamics and kinetics of the alpha-spectrin SH3 domain with a redesigned hydrophobic core have been studied. The introduction of five replacements, A11V, V23L, M25V, V44I and V58L, resulted in an increase of 16% in the overall volume of the side-chains forming the hydrophobic core but caused no remarkable changes to the positions of the backbone atoms. Judging by the scanning calorimetry data, the increased stability of the folded structure of the new SH3-variant is caused by entropic factors, since the changes in heat capacity and enthalpy upon the unfolding of the wild-type and mutant proteins were identical at 298 K. It appears that the design process resulted in an increase in burying both the hydrophobic and hydrophilic surfaces, which resulted in a compensatory effect upon the changes in heat capacity and enthalpy. Kinetic analysis shows that both the folding and unfolding rate constants are higher for the new variant, suggesting that its transition state becomes more stable compared to the folded and unfolded states. The phi(double dagger-U) values found for a number of side-chains are slightly lower than those of the wild-type protein, indicating that although the transition state ensemble (TSE) did not change overall, it has moved towards a more denatured conformation, in accordance with Hammond's postulate. Thus, the acceleration of the folding-unfolding reactions is caused mainly by an improvement in the specific and/or non-specific hydrophobic interactions within the TSE rather than by changes in the contact order. Experimental evidence showing that the TSE changes globally according to its hydrophobic content suggests that hydrophobicity may modulate the kinetic behaviour and also the folding pathway of a protein.     

Thermodynamics of core hydrophobicity and packing in the hyperthermophile proteins Sac7d and Sso7d

The influence of core hydrophobicity and packing on the structure and stability of the hyperthermophile proteins Sac7d and Sso7d have been studied by calorimetry, circular dichroism, and NMR. Valine 30 is positioned in Sac7d to allow a cavity-filling Val --> Ile substitution which occurs naturally in the homologous more thermostable Sso7d. The cavity-filling mutation in Sac7d has been characterized and compared to the reciprocal Ile --> Val mutation in Sso7d. A detailed analysis of the stability of the proteins was obtained by globally fitting the variation of DSC parameters and circular dichroism intensities as a function of temperature (0-100 degrees C), salt (0-0.3 M), and pH (0-8). A global analysis over such a range of conditions permitted an unusually precise measure of the thermodynamic parameters, as well as the separation of the thermodynamics of the intrinsic unfolding reaction from the linked effects of protonation and chloride binding associated with acid-induced folding. The results indicate differences in the energetics of unfolding Sac7d and Sso7d that would not be apparent from an analysis of DSC data alone using conventional methods. The sign and magnitude of the changes in DeltaG, DeltaH, TDeltaS, and DeltaC(P) of unfolding resulting from core Ile/Val substitutions in the two proteins were consistent with differences in hydrophobicity of Val and Ile and negligible changes in packing (van der Waals) interactions. The benefit of increased hydrophobicity of the core increased with temperature, with maximal effect around 116 degrees C. Increased hydrophobicity of the core achieved not only an increase in the free energy of unfolding, but also a lateral shift of the temperature of maximal stability to higher temperature.     

Mutations in the hydrophobic core and in the protein-RNA interface affect the packing and stability of icosahedral viruses.

The information required for successful assembly of an icosahedral virus is encoded in the native conformation of the capsid protein and in its interaction with the nucleic acid. Here we investigated how the packing and stability of virus capsids are sensitive to single amino acid substitutions in the coat protein. Tryptophan fluorescence, bis-8-anilinonaphthalene-1-sulfonate fluorescence, CD and light scattering were employed to measure urea- and pressure-induced effects on MS2 bacteriophage and temperature sensitive mutants. M88V and T45S particles were less stable than the wild-type forms and completely dissociated at 3.0 kbar of pressure. M88V and T45S mutants also had lower stability in the presence of urea. We propose that the lower stability of M88V particles is related to an increase in the cavity of the hydrophobic core. Bis-8-anilinonaphthalene-1-sulfonate fluorescence increased for the pressure-dissociated mutants but not for the urea-denatured samples, indicating that the final products were different. To verify reassembly of the particles, gel filtration chromatography and infectivity assays were performed. The phage titer was reduced dramatically when particles were treated with a high concentration of urea. In contrast, the phage titer recovered after high-pressure treatment. Thus, after pressure-induced dissociation of the virus, information for correct reassembly was preserved. In contrast to M88V and T45S, the D11N mutant virus particle was more stable than the wild-type virus, in spite of it also possessing a temperature sensitive growth phenotype. Overall, our data show how point substitutions in the capsid protein, which affect either the packing or the interaction at the protein-RNA interface, result in changes in virus stability.     

Role of the charge-charge interactions in defining stability and halophilicity of the CspB proteins

Charge-charge interactions on the surface of native proteins are important for protein stability and can be computationally redesigned in a rational way to modulate protein stability. Such computational effort led to an engineered protein, CspB-TB that has the same core as the mesophilic cold shock protein CspB-Bs from Bacillus subtilis, but optimized distribution of charge-charge interactions on the surface. The CspB-TB protein shows an increase in the transition temperature by 20 degrees C relative to the unfolding temperature of CspB-Bs. The CspB-TB and CspB-Bs protein pair offers a unique opportunity to further explore the energetics of charge-charge interactions as the substitutions at the same sequence positions are done in largely similar structural but different electrostatic environments. In particular we addressed two questions. What is the contribution of charge-charge interactions in the unfolded state to the protein stability and how amino acid substitutions modulate the effect of increase in ionic strength on protein stability (i.e. protein halophilicity). To this end, we experimentally measured the stabilities of over 100 variants of CspB-TB and CspB-Bs proteins with substitutions at charged residues. We also performed computational modeling of these protein variants. Analysis of the experimental and computational data allowed us to conclude that the charge-charge interactions in the unfolded state of two model proteins CspB-Bs and CspB-TB are not very significant and computational models that are based only on the native state structure can adequately, i.e. qualitatively (stabilizing versus destabilizing) and semi-quantitatively (relative rank order), predict the effects of surface charge neutralization or reversal on protein stability. We also show that the effect of ionic strength on protein stability (protein halophilicity) appears to be mainly due to the screening of the long-range charge-charge interactions.     

Pressure versus temperature unfolding of ribonuclease A: an FTIR spectroscopic characterization of 10 variants at the carboxy-terminal site

FTIR spectroscopy was used to characterize and compare the temperature- and pressure-induced unfolding of ribonuclease A and a set of its variants engineered in a hydrophobic region of the C-terminal part of the molecule postulated as a CFIS. The results show for all the ribonucleases investigated, a cooperative, two-state, reversible unfolding transition using both pressure and temperature. The relative stabilities, among the different sites and different variants at the same site, monitored either through the changes in the position of the maximum of the amide I' band and the tyrosine band, or the maximum of the band assigned to the beta-sheet structure, corroborate the results of a previous study using fourth-derivative UV absorbance spectroscopy. In addition, variants at position 108 are the most critical for ribonuclease structure and stability. The V108G variant seems to present a greater conformational flexibility than the other variants. The pressure- and temperature-denaturated states of all the ribonucleases characterized retained some secondary structure. However, their spectral maxima were centered at different wavenumbers, which suggests that pressure- and temperature-denaturated states do not have the same structural characteristics. Nevertheless, there was close correlation between the pressure and temperature midpoint transition values for the whole series of protein variants, which indicated a common tendency of stability toward pressure and heat.     

Polar residues in the protein core of Escherichia coli thioredoxin are important for fold specificity

Most globular proteins contain a core of hydrophobic residues that are inaccessible to solvent in the folded state. In general, polar residues in the core are thermodynamically unfavorable except when they are able to form intramolecular hydrogen bonds. Compared to hydrophobic interactions, polar interactions are more directional in character and may aid in fold specificity. In a survey of 263 globular protein structures, we found a strong positive correlation between the number of polar residues at core positions and protein size. To probe the importance of buried polar residues, we experimentally tested the effects of hydrophobic mutations at the five polar core residues in Escherichia coli thioredoxin. Proteins with single hydrophobic mutations (D26I, C32A, C35A, T66L, and T77V) all have cooperative unfolding transitions like the wild type (wt), as determined by chemical denaturation. Relative to wt, D26I is more stable while the other point mutants are less stable. The combined 5-fold mutant protein (IAALV) is less stable than wt and has an unfolding transition that is substantially less cooperative than that of wt. NMR spectra as well as amide deuterium exchange indicate that IAALV is likely sampling a number of low-energy structures in the folded state, suggesting that polar residues in the core are important for specifying a well-folded native structure.