Stability Of Mutant Proteins Research Articles

Computational studies on mutant protein stability: The correlation between surface thermal expansion and protein stability.

Thermal stability of mutant proteins has been investigated using temperature dependent molecular dynamics (MD) simulations in vacuo. The numerical modeling was aimed at mimicking protein expansion upon heating. After the conditions for an expanding protein accessible surface area were established for T4 lysozyme and barnase wild-type proteins, MD simulations were carried out under the same conditions using the crystal structures of several mutant proteins. The computed thermal expansion of the accessible surface area of mutant proteins was found to be strongly correlated with their experimentally measured stabilities. A similar, albeit weaker, correlation was observed for model mutant proteins. This opens the possibility of obtaining stability information directly from protein structure.

New evidence for the denaturant binding model.

Denaturation with guanidine hydrochloride (GdnHCl) or urea is one of the primary ways of measuring the conformational stability of proteins and comparing the stability of mutant proteins. Despite the widespread use of these two denaturants to provide quantitative data for the free energies of unfolding, the mode of action of these agents is not well understood. We are not even certain whether the action of these agents on proteins is direct and can be regarded as ligand binding, or indirect and involves a change in the properties of solvent (water) in the presence of GdnHCl and urea. In this paper, an extensive kinetic study of the inhibition of ribonuclease A and papain by urea has been performed. The results suggest that the effect of urea on activities of these enzymes can be well described by the denaturant binding model. The binding constants of urea determined by the present method are nearly identical to that determined from a variety of different studies on model compounds and proteins.

Genetic analysis of the large subunit of yeast transcription factor IIE reveals two regions with distinct functions.

Biochemical analysis of proteins necessary for transcription initiation by eukaryotic RNA polymerase II (pol II) has identified transcription factor IIE (TFIIE) as an essential component of the reaction. To better understand the role of TFIIE in transcription, we isolated conditional alleles of TFA1, the gene encoding the large subunit of TFIIE in the yeast Saccharomyces cerevisiae. The mutant Tfa1 proteins fall into two classes. The first class causes thermosensitive growth due to single amino acid substitutions of the cysteines comprising the Zn-binding motif. The second mutant class is made up of proteins that are C-terminally truncated and that cause a cold-sensitive growth phenotype. The behavior of these mutants suggests that Tfa1p possesses at least two domains with genetically distinct functions. The mutations in the Zn-binding motif do not affect the mutant protein's stability at the nonpermissive temperature or its ability to associate with the small subunit of TFIIE. Our studies further determined that wild-type TFIIE can bind to single-stranded DNA in vitro. However, this property is unaffected in the mutant TFIIE complexes. Finally, we have demonstrated the biological importance of TFIIE in pol II-mediated transcription by depleting the Tfa1 protein from the cells and observing a concomitant decrease in total poly(A)+ mRNA.

Contributions of the ionizable amino acids to the stability of staphylococcal nuclease.

To quantitate the contributions of the ionizable amino acids to the stability of the native state of staphylococcal nuclease, each of the 23 lysines, 5 arginines, 4 histidines, 12 glutamic acids, and 8 aspartic acids was substituted with both alanine and glycine. This collection of 104 mutant proteins was analyzed by guanidine hydrochloride (GuHCl) denaturation, using intrinsic tryptophan fluorescence to quantitate the equilibrium between native and denatured states. From the analysis of these data, each mutant protein's stability in the absence of denaturant (delta GH2O) and sensitivity to changes in denaturant concentration [mGuHCl = d(delta G)/d[GuHCl]] were obtained. Several general trends in these values suggest that electrostatic interactions make only a minor contribution to the net stability of this protein. For the residue pairs that form ten salt bridges and ten charged hydrogen bonds between side chains, no correlation was observed between the stability losses (delta delta G) accompanying alanine substitution of each member of the pair. Little or no significant correlation was found between the magnitude of the loss in stability and the local electrostatic potential calculated from the three-dimensional structure by numerical and model dependent solutions of the linearized Poisson-Boltzmann equation. The structural parameters which correlated most strongly with stability loss are measures of the extent of burial of the residue in the native structure, as was previously observed for alanine and glycine substitutions of large hydrophobic residues [Shortle et al. (1990) Biochemistry 29, 8033] and of the polar, uncharged residues [Green et al. (1992) Biochemistry 31, 5717]. These results suggest that the ionizable amino acids contribute to stability predominantly through packing and bonding interactions that do not depend on their electrostatic charge.

Structural and genetic analysis of protein stability.

One very encouraging development has been the freedom with which amino acid replacements can be introduced in a protein of interest. This has made it possible to obtain detailed structural and thermodynamic data on a wide variety of mutants that modify protein stability. Substitutions of solvent-exposed amino acids on the surfaces of proteins are seen to have little if any effect on protein stability or structure, leading to the view that it is the rigid parts of proteins that are critical for folding and stability. There is every reason to expect that it will be possible to rationalize the stabilities of mutant proteins from accurate knowledge of their structures. Substantial progress is being made in quantitating the interactions that determine and stabilize protein structures. Although not specifically the subject of this review, substantial progress is also being made in developing methods to engineer proteins of enhanced stability.

Extracting hydrophobic free energies from experimental data: relationship to protein folding and theoretical models

Solubility and vapor pressure measurements of hydrocarbons in water are generally thought to provide estimates of the strength of the hydrophobic effect in the range 20-30 cal/(mol.A2). Our reassessment of the solubility data on the basis of new developments in solution thermodynamics suggests that the hydrophobic surface free energy for hydrocarbon solutes is 46-47 cal/(mol.A2), although the actual value depends strongly on curvature effects [Nicholls et al. (1991) Proteins (in press); Sharp et al. (1991) Science 252, 106-109]. The arguments to support such a significant increase in the estimate of the hydrophobic effect stem partly from theoretical considerations and partly from the experimental results of De Young and Dill [(1990) J. Phys. Chem. 94, 801-809] on benzene partition between water and alkane solvents. Previous estimates of the hydrophobic effect derive from an analysis of solute partition data, which does not fully account for changes in volume entropy. We show here how the ideal gas equations, combined with experimental molar volumes, can account for such changes. Revised solubility scales for the 20 amino acids, based on cyclohexane to water and octanol to water transfer energies, are derived. The agreement between these scales, particularly the octanol scale, and mutant protein stability measurements from Kellis et al. [(1989) Biochemistry 28, 4914-4922] and Shortle et al. [(1990) Biochemistry 29, 8033-8041] is good. The increased strength of the hydrophobic interaction has implications for the energetics of protein folding, substrate binding, and nucleic acid base stacking and the interpretation of computer simulations.

Conformational stability of pig citrate synthase and some active-site mutants

The conformational stabilities of native pig citrate synthase (PCS), a recombinant wild-type PCS, and six active-site mutant pig citrate synthases were studied in thermal denaturation experiments by circular dichroism and in urea denaturation experiments by using DTNB to measure the appearance of latent SH groups. His274 and Asp375 are conserved active-site residues in pig citrate synthase that bind to substrates and are implicated in the catalytic mechanism of the enzyme. By site-directed mutagenesis, His274 was replaced with Gly and Arg, while Asp375 was replaced with Gly, Asn, Glu, or Gln. These modifications were previously shown to result in 10(3)-10(4)-fold reductions in enzyme specific activities. The thermal unfolding of pig citrate synthase and the six mutants in the presence and absence of substrates showed large differences in the thermal stabilities of mutant proteins compared to the wild-type pig citrate synthase. The functions of His274 and Asp375 in ligand binding were measured by oxalacetate protection against urea denaturation. These data indicate that active-site mutations that decrease the specific activity of pig citrate synthase also cause an increase in the conformational stability of the protein. These results suggest that specific electrostatic interactions in the active site of citrate synthase are important in the catalytic mechanism in the chemical transformations as well as the conformational flexibility of the protein, both of which are important for the overall catalytic efficiency of the enzyme.

Contributions of the large hydrophobic amino acids to the stability of staphylococcal nuclease

To quantitate the contributions of the large hydrophobic residues in staphylococcal nuclease to the stability of its native state, single alanine and glycine substitutions were constructed by site-directed mutagenesis for each of the 11 leucine, 9 valine, 7 tyrosine, 5 isoleucine, 4 methionine, and 3 phenylalanine residues. In addition, each isoleucine was also mutated to valine. The resulting collection of 83 mutant nucleases was submitted to guanidine hydrochloride denaturation using intrinsic tryptophan fluorescence to monitor the equilibrium constant between the native and denatured states. From analysis of these data, each mutant protein's stability to reversible denaturation (delta GH2O) and sensitivity to guanidine hydrochloride (mGuHCl or d(delta G)/d[GuHCl]) were obtained. Four unexpected trends were observed. (1) A striking bipartite distribution was found for sites of mutations that altered mGuHCl: mutations that increased this parameter only involved residues that contribute side chains to the major hydrophobic core centered around a five-strand beta-barrel, whereas mutations that caused mGuHCl to decrease clustered around a second, smaller and less well-defined hydrophobic core. (2) The average stability loss for mutants in each of the six residue classes was 2-3 times greater than that estimated on the basis of the free energy of transfer of the hydrophobic side chain from water to n-octanol. (3) The magnitude of the stability loss on substituting Ala or Gly for a particular type of amino acid varied extensively among the different sites of its occurrence in nuclease, indicating that the environment surrounding a specific residue determines how large a stability contribution its side chain will make.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrolyte effects on the activity of mutant enzymes in vivo and in vitro

All temperature-sensitive histidine auxotrophs of Salmonella typhimurium tested are corrected by addition of neutral salts to their growth medium. The correctability seems to result from direct electrolyte effects on mutant protein stability since several of the mutant proteins are also salt correctable in vitro.