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W E Stites

Publications and source records attributed to W E Stites.

22 records · Page 2Linked to original sources
PubMed1993

The phase transition between a compact denatured state and a random coil state in staphylococcal nuclease is first-order.

Three mutants of staphylococcal nuclease containing a tryptophan substitution have been examined in the full length (149 residues) protein and in a large fragment (residues 1 to 136). The large fragments are not in the native state and are a good model of the denatured state. However, these large fragments do show signs of residual structure that breaks down upon titration with guanidine hydrochloride. They share some similarities with what has become known as the molten globule state. The thermal unfolding of these mutant fragments was followed by tryptophan fluorescence. Tryptophan fluorescence was treated as an order parameter and analyzed to determine the order of the observed transition. The critical exponent of the order parameter as the transition temperature is approached is significantly higher than the value of 1/2 predicted by mean field theory for a second-order transition and is similar to that observed for the transition of the full length, wild-type, protein. This is strong evidence that the breakdown of this intermediate compact denatured state is a cooperative, first-order phenomenon.

Micrococcal Nuclease↗
PubMed1991

In a staphylococcal nuclease mutant the side-chain of a lysine replacing valine 66 is fully buried in the hydrophobic core.

The crystal structure of the staphylococcal nuclease mutant V66K, in which valine 66 is replaced by lysine, has been solved at 1.97 A resolution. Unlike lysine residues in previously reported protein structures, this residue appears to bury its side-chain in the hydrophobic core without salt bridging, hydrogen bonding or other forms of electrostatic stabilization. Solution studies of the free energy of denaturation, delta GH2O, show marked pH dependence and clearly indicate that the lysine residue must be deprotonated in the folded state. V66K is highly unstable at neutral pH but only modestly less stable than the wild-type protein at high pH. The pH dependence of stability for V66K, in combination with similar measurements for the wild-type protein, allowed determination of the pKa values of the lysine in both the denatured and native forms. The epsilon-amine of this residue has a pKa value in the denatured state of 10.2, but in the native state it must be 6.4 or lower. The epsilon-amine is thus deprotonated in the folded molecule. These values enabled an estimation of the epsilon-amine's relative change in free energy of solvation between solvent and the protein interior at 5.1 kcal/mol or greater. This implies that the value of the dielectric constant of the protein interior must be less than 12.8. Lysine is usually found with the methylene groups of its side-chain partly buried but is nevertheless considered a hydrophilic surface residue. It would appear that the high pKa value of lysine, which gives it a positive charge at physiological pH, is the primary reason for its almost exclusive confinement to the surface proteins. When deprotonated, this amino acid type can be fully incorporated into the hydrophobic core.

Computer Simulation↗
PubMed1990

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)

Amino Acids↗