GSBS Student Publications

Title

Systematic mutation of bacteriophage T4 lysozyme

UMMS Affiliation

Graduate School of Biomedical Sciences; Department of Molecular Genetics and Microbiology

Date

11-5-1991

Document Type

Article

Medical Subject Headings

Amino Acid Sequence; Base Sequence; DNA, Viral; Models, Molecular; Molecular Sequence Data; Muramidase; *Mutagenesis; Phenotype; Plasmids; Proline; Suppression, Genetic; T-Phages; Temperature

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).

Rights and Permissions

Citation: J Mol Biol. 1991 Nov 5;222(1):67-88.

Related Resources

Link to Article in PubMed

Journal Title

Journal of molecular biology

PubMed ID

1942069