Amino-acid substitutions at the fully exposed P-1 site of bovine pancreatic trypsin inhibitor affect its stability

Krowarsch, D Otlewski, J

D. Krowarsch et J. Otlewski, Amino-acid substitutions at the fully exposed P-1 site of bovine pancreatic trypsin inhibitor affect its stability, PROTEIN SCI, 10(4), 2001, pp. 715-724

54

INGLESE

Article

Biochemistry & Biophysics

PROTEIN SCIENCE

0961-8368 → ACNP

10

4

2001

715 - 724

ISI

0961-8368(200104)10:4<715:ASATFE>2.0.ZU;2-M

It is widely accepted that solvent-exposed sites in proteins play only a ne glible role in determining protein energetics. In this paper we show that a mino acid substitutions at the fully exposed tys15 in bovine pancreatic try psin 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) m utant 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) diffe rence of 2.1 kcal . mole(-1). Thermal denaturation of the variants monitore d by CD signal at pH 2.0 in the presence of 6 M GdmCl again showed differen ces 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 differ ences. A correlation analysis of the P-1 stability with different propel ti es of amino acids suggests that two mechanisms may be responsible for the o bserved stability differences: the reverse hydrophobic effect and amino aci d propensities to occur in nonoptimal dihedral angles adopted by the P-1 po sition. The former effect operates at the denatured state level and causes a drop in protein stability for hydrophobic side chains, due to their decre ased exposure upon denaturation. The latter factor influences the native st ate energetics and results from intrinsic properties of amino acids in a wa y similar to those observed for secondary structure propensities. In conclu sion, our results suggest that the protein-stability-derived secondary stru cture propensity scales should be taken with more caution.