An Investigation of the Savinase Water Channel: Implications of Cavity Mutations

Abstract

The interior packing of proteins is believed to play a crucial role in stabilizing a protein’s conformation. Recently, the influence of changes in the interior packing on thermostability and function of proteins have been examined by means of protein engineering. Hydrophobic residues were substituted by more bulky residues in T4 lysozyme to fill a large cavity^1,2 in order to improve the interior packing. Crystal structure analysis of the mutant proteins showed that the introduced aminoacids are accommodated with little perturbation of the three dimensional structures. The mutant proteins had normal activities and their thermal stabilities were marginally lower than those of the wildtype protein. Although the incorporation of more bulky hydrophobic sidechains in the core is expected to result in an increase in hydrophobic (entropic) stabilization a reduction in stability was observed. This result was ascribed to introduction of strain in the form of non-optimal dihedral angles, bond angle distortion and unfavorable van der Waals contacts (a positive free energy contribution).