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Systematic mutation of bacteriophage T4 lysozyme
D Rennell1, S E Bouvier, L W Hardy
1Department of Molecular Genetics and Microbiology, University of Massachusetts, Worcester.
Journal of Molecular Biology
|November 5, 1991
Summary
Amber mutations in bacteriophage T4 lysozyme revealed critical residues. Many protein positions tolerate substitutions, but specific sites are essential for lysozyme function and structure.
Area of Science:
- Molecular Biology
- Protein Engineering
- Virology
Background:
- Bacteriophage T4 lysozyme is essential for phage P22 plaque formation.
- Amber mutations are powerful tools for probing protein function by introducing specific amino acid substitutions.
Purpose of the Study:
- To systematically map the functional landscape of T4 lysozyme by assessing the impact of single amino acid substitutions at nearly every position.
- To identify residues critical for T4 lysozyme activity and structural integrity.
Main Methods:
- Generated a comprehensive library of amber mutants across the T4 lysozyme gene.
- Tested plaque formation of these mutants on Salmonella typhimurium strains expressing suppressor tRNAs.
- Quantified the impact of mutations on lysozyme activity relative to wild-type.
Main Results:
- Over 2000 single amino acid substitutions were analyzed.
- 328 substitutions were found to be deleterious, inhibiting plaque formation.
- 55% of T4 lysozyme positions tolerated all tested substitutions, indicating functional robustness.
Conclusions:
- Identified specific residues crucial for T4 lysozyme activity and structural stability, including catalytic and buried salt bridge residues.
- Demonstrated that solvent accessibility is a general predictor of residue tolerance to mutation, with exceptions.
- Provided insights into structure-function relationships within T4 lysozyme.