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

Laboratory of Protein Engineering, Institute of Biochemistry and Molecular Biology, University of Wroclaw, 50–137 Wroclaw, Poland

Reprint requests to: Jacek Otlewski, Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50–137 Wroclaw, Poland; e-mail: otlewski{at}bf.uni.wroc.pl fax: (48) 71 3752608.

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 Lys15 in bovine pancreatic trypsin inhibitor (BPTI) influenced the CD- and DSC-monitored stability: The T_den difference between the least (P₁ Trp) and the most stable (P₁ His) mutant is 11.2°C at pH 2.0. The H_den versus T_den plot for all the variants at three pH values (2.0, 2.5, 3.0) is linear (C_p,den = 0.41 kcal• mole^-1 • K^-1; 1 cal = 4.18 J) leading to a G_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 (T_den =7.1°C). Selective reduction of the Cys14–Cys 38 disulfide bond, which is located in the vicinity of the P₁ position did not eliminate the stability differences. A correlation analysis of the P₁ stability with different properties 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 P₁ 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.

Keywords: Thermodynamic stability; solvent-exposed residue; reverse hydrophobic effect; bovine pancreatic trypsin inhibitor

Abbreviations: BPTI, bovine pancreatic trypsin inhibitor • OMTKY3, turkey ovomucoid third domain • SSI, Streptomyces subtilisin inhibitor • CD, circular dichroism • DSC, differential scanning calorimetry • GdmCl, guanidinium chloride • H_cal, calorimetric enthalpy • H_vH, van't Hoff enthalpy • S_den, H_den, C_p,den, and G_den, entropy, enthalpy, heat capacity, and free-energy changes, respectively, accompanying protein denaturation • T_den, denaturation temperature

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