Isolation and characterization of multiple-methionine mutants of T4 lysozyme with simplified cores

Abstract

Publisher Summary This chapter describes the degree to which precise spatial complementarity among core residues is required to maintain native-like protein properties. Sites within the carboxy-terminal domain core of phage T4 lysozyme has been substituted singly and as a group with methionine to produce a simplified core sequence. The properties of such mutant lysozymes are briefly described. In addition, the chapter describes a method to isolate mutant protein from inclusion bodies and a sensitive enzymatic assay to detect small differences in mutant protein activities. The carboxy-terminal domain of T4 lysozyme is composed of seven helices and includes the largest contiguous set of buried residues in the protein. Side-chains are considered a part of the core if they have less than 10% solvent accessible surface. The carboxy-terminal domain also contains a single, completely buried methionine (Met 102), and two others (Met 106 and Met 120), the side-chains of which are about 80% buried. The fact that at least seven core residues can be replaced as a group with methionine in phage T4 lysozyme without introducing molten globule-like characteristics shows that strict side-chain complementarity is not required to maintain native-like protein properties. The crystallographic thermal factors of the side-chains of some of the seven methionines in the mutant structure are slightly higher than the wild-type amino acids they replace. The thermal factors of the three methionines that are present within the carboxy-terminal domains of both structures are, if anything, better ordered in the mutant molecule. In all cases, the electron density for the introduced methionines is well defined. There is no suggestion that substitution of seven methionines leads to disorder within the protein core or in other regions of the structure.