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

Abstract

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)

Interactions in nonnative and truncated forms of staphylococcal nuclease as indicated by mutational free energy changes

“…The mden value asymptotically decreases with increased hydrophobicity. Changes in mden have previously been observed in conventional mutagenesis experiments at position 23 in nuclease (Shortle et al, 1990). The change in mden values, under the Schellman theory discussed above, suggests decreased surface area in the nonnative state(s) of nuclease as the hydrophobicity is increased.…”

“…Changes in m&fl have been observed for a large number of variants of staphylococcal nuclease (Shortle & Meeker, 1986;Green et al, 1992). Based on the correlation between changes in mden values and changes in stability, Shortle and colleagues have concluded that amino acid changes can affect the structure and free energy of the denatured state (Shortle et al, 1990). It is also possible to account for changes in mden by postulating a three-state mechanism ( N + + I -U).…”

Chemically crosslinked protein dimers: Stability and denaturation effects

“…However, there is a more relevant interpretation of changes in slope value. It has been postulated that changes in mGuHCl can be attributed to changes in the amount of solventaccessible hydrophobic surface area exposed during the transition between the native and denatured states (Schellman, 1978;Shortle et al, 1990;Dill & Shortle, 1991). According to this interpretation, higher slope values are found in those proteins that expose more hydrophobic surface area in the denatured state relative to the amount exposed in the native state.…”

“…The data was treated as a two-state model for reversible denaturation in which each protein molecule exists as folded or unfolded, whereas intermediate states are only transiently populated. Data analysis was carried out as previously published (Shortle et al, 1990;Stites et al, 1995). This analysis yields three parameters: a protein's stability to reversible denaturation (AGHZ0), the rate of change of free energy with respect to GuHCl concentration (rnGuHCI or d(AG)/d [GuHCI]), and the concentration of guanidine hydrochloride at which half the protein molecules are denatured (C,,,).…”

P100, a transcriptional coactivator, is a human homologue of staphylococcal nuclease

“…Conserved hydrophobic and/or small residues (Fig. I ) are known to contribute to S. aureus SNase protein stability (Shortle et al, 1990;Green et a!., 1992). Variants containing substitutions of Gly-107 or Ala-I32 are among the most unstable of SNase mutants (Green et a!., 1992), and it is notable that these are among the most conserved residues of SNase homologues, including those in pl00 orthologues (Fig.…”