Crevice‐forming mutants of bovine pancreatic trypsin inhibitor: Stability changes and new hydrophobic surface

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Abstract
Four mutants of bovine pancreatic trypsin inhibitor (BPTI) with replacements in the rigid core result in the creation of deep crevices on the surface of the protein. Other than crevices at the site of the mutation, few other differences are observed in the crystal structures of wild‐type BPTI and the mutants F22A, Y23A, N43G, and F45A. These mutants are highly destabilized relative to wild type (WT). The differences between WT and mutants in the free energy change associated with cooperative folding/unfolding, ΔΔG0(WT→mut), have been measured by calorimetry, and they are in good agreement with ΔΔG0(WT→mut) values from hydrogen exchange rates. For F22A the change in free energy difference is about 1.7 kcal/mol at 25 °C; for the other three mutants it is in the range of 5–7 kcal/mol at 25 °C. The experimental ΔΔG0(WT→mut) values of F22A, Y23A, and F45A are reasonably well accounted for as the sum of two terms: the difference in transfer free energy change, and a contribution from exposure to solvent of new surface (Eriksson, A.E., et al., 1992, Science 255, 178–183), if the recently corrected transfer free energies and surface hydrophobicities (De Young, L. & Dill, K., 1990, J. Phys. Chem. 94, 801–809; Sharp, K.A., et al., 1991a, Science 252, 106–109) are used and only nonpolar surface is taken into account. In N43G, three protein–protein hydrogen bonds are replaced by protein–water hydrogen bonds. The value of ΔΔG0(WT→mut) computed from model transfer free energies and surface hydrophobicities is 4.0 kcal/mol less than observed, consistent with reports that one hydrogen bond contributes about 1.3 kcal/mol of stabilizing free energy (Shirley, B.A., et al., 1992, Biochemistry 31, 725–732).
Funding Information
  • NIH (GM26246)
  • National Cancer Institute, DHHS (N01-CO-74101)