Amino-acid substitutions at the fully exposed P1 site of bovine pancreatic trypsin inhibitor affect its stability

It is widely accepted that solvent-exposed sites in proteins play only a neglible role in determining protein energetics. In this paper we show that amino acid substitutions at the fully exposed tys15 in bovine pancreatic trypsin inhibitor (BPTI) influenced the CD- and DSC-monitored stability: The T-den difference between the least (P-1 Trp) and the most stable (P-1 His) mutant is 11.2 degreesC at pH 2.0, The DeltaH(den) versus T-den plot for all the variants at three pH values (2.0, 2.5, 3.0) is linear (DeltaC(p,den) = 0.41 kcal . mole(-1) . K-1; 1 cal = 4.18 J) leading to a DeltaG(den) difference of 2.1 kcal . mole(-1). Thermal denaturation of the variants monitored by CD signal at pH 2.0 in the presence of 6 M GdmCl again showed differences in their stability, albeit somewhat smaller (DeltaT(den) = 7.1 degreesC). Selective reduction of the Cys14-Cys 38 disulfide bond, which is located in the vicinity of the P-1 position did not eliminate the stability differences. A correlation analysis of the P-1 stability with different propel ties of amino acids suggests that two mechanisms may be responsible for the observed stability differences: the reverse hydrophobic effect and amino acid propensities to occur in nonoptimal dihedral angles adopted by the P-1 position. The former effect operates at the denatured state level and causes a drop in protein stability for hydrophobic side chains, due to their decreased exposure upon denaturation. The latter factor influences the native state energetics and results from intrinsic properties of amino acids in a way similar to those observed for secondary structure propensities. In conclusion, our results suggest that the protein-stability-derived secondary structure propensity scales should be taken with more caution.