Alteration of T4 lysozyme structure by second‐site reversion of deleterious mutations

Abstract

Mutations that suppress the defects introduced into T4 lysozyme by single amino acid substitutions were isolated and characterized. Among 53 primary sites surveyed, 8 yielded second-site revertants; a total of 18 different mutants were obtained. Most of the restorative mutations exerted global effects, generally increasing lysozyme function in a number of primary mutant contexts. Six of them were more specific, suppressing only certain specific deleterious primary substitutions, or diminishing the function of lysozymes bearing otherwise nondeleterious primary substitutions. Some variants of proteins bearing primary substitutions at the positions of Asp 20 and Ala 98 are inferred to have significantly altered structures.Keywords: bacteriophage; mutations; second-site revertants; T4 lysozymeThe lysozyme encoded by bacteriophage T4 is a small, globular, monomeric protein that has been the subject of extensive structural and genetic studies (for reviews, see Potkte & Hardy, 1994;Matthews, 1995). The native structure of the protein has been determined to 1.7 8, resolution by X-ray crystallography (Weaver & Matthews, 1987).Systematic probing of the T4 lysozyme structure has shown that 74 of the 164 positions in its sequence are sensitive to single amino acid substitutions; that is, at least one single amino acid substitution at one of these positions results in at least a 50-fold reduction in function (Rennell et al., 1991). Not surprisingly, the most critical amino acid residues in the protein were found to be either buried, or else solvent-exposed, but in the active site cleft.A second level of genetic probing of protein structure consists of isolating and characterizing intragenic second-site revertants of mutant proteins. A survey of second-site suppressors could be expected to reveal interactions between residues in the folded (or possibly, folding) structure. Some of the resulting multiply mutant proteins might be expected to have significantly altered structures, the solution of which could lead to insights on the protein folding problem.An early study of second-site revertants of temperature-sensitive mutants of staphylococcal nuclease (Shortle & Lin, 1985) Bouvier & Poteete, 1996) uncovered structural variants in addition to global stabilizers.Here we report the results of a systematic survey of second-site revertants in the T4 lysozyme gene. Presumptively significant structural variants are described. ResultsIn a previous study (Rennell et al., 1991), we examined the effects of single amino acid substitutions in the T4 lysozyme gene by the use of amber suppressors. A bacteriophage P22 hybrid bearing the T4 lysozyme gene in place of its P22 counterpart was constructed, amber mutations were introduced into its lysozyme gene, and the mutant phages were tested for ability to form plaques on a collection of 13 Salmonella amber suppressor strains that each insert a different amino acid in response to the amber codon. Of 163 amber mutant phages tested in this way, 74 failed to form plaques, or formed tiny p...

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