THE RESPONSE OF T4 LYSOZYME TO LARGE-TO-SMALL SUBSTITUTIONS WITHIN THE CORE AND ITS RELATION TO THE HYDROPHOBIC EFFECT

To further examine the structural and thermodynamic basis of hydrophob ic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were subs tituted with alanine. In 25 cases, including eight reported previously , it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also d etermined. In the majority of cases the ''large-to-small'' substitutio ns lead to internal cavities. In other cases declivities or channels o pen to the surface were formed. In some cases the structural changes w ere minimal (mainchain shifts less than or equal to 0.3 Angstrom); in other cases mainchain atoms moved up to 2 Angstrom. In the case of Ile 29 --> Ala the structure collapsed to such a degree that the volume o f the putative cavity was zero. Crystallographic analysis suggests tha t the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 --> Ala (V149A) and Met 6 --> Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the cavity. The Val 149 --> Ala substitution allows t he solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with al anine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 --> Ala the loss of stability of all the cavity mutants can be rationalized as a combi nation of two terms. The first is a constant for a given class of subs titution (e.g., -2.1 kcal/mol for all Leu --> Ala substitutions) and c an be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The s econd term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol(-1) Angstrom(-3 ). Physically, this term is due to the loss of van der Waal's interact ions between the bulky sidechain that is removed and the atoms that fo rm the wall of the cavity. The overall results are consistent with the prior rationalization of Leu --> Ala mutants in T4 lysozyme by Erikss on et al. (Eriksson et al., 1992, Science 255:178-183).