A Genetic and Structural Analysis of P22 Lysozyme: A Thesis

GENETIC AND STRUCTURAL ANAYLYSIS OF P22 LYSOZYME P22 lysozyme , encoded by gene 19, is an essential phage protein responsible for hydrolyzing the bacterial cell wall during lytic infection. P22 lysozyme is related to T4 lysozyme in its mode of action, substrate specificities , and in its structure. Gene 19 was located on the phage genome , subcloned and then sequenced. Lysozyme was produced in large quanti ties and purified for biochemical characterization and for crystallograpic studies. Gene 19 consists of 146 codons , and encodes a protein with a molecular weight of 16 117. Amer mutations were created in gene 19 by in vitro primer-directed mutagenesis. The mutations were crossed by homologous recombination onto the phage genome. The phages bearing the amber mutations in gene 19 were screened for the ability to grow on six different amber suppressor strains. Amino acid substitutions that resulted in nonfunctional or less functional lysozyme were determined. Of 60 possible amino acid substitutions at 11 different sites in P22 lysozyme , 20 are deleterious. The phage bearing amber mutations in gene 19 that failed to grow on given suppressor strains were reverted and second site intragenic revertants were obtained. The mutations were sequenced. A substitution of serine for glutamine at residue 82 is compensated for by changing residue 46 from serine to leucine. This single change enables the phage to form a plaque at 30 C but not at 40 C. When the triple change asn42lys; ser46leu; and ser43pro is present the lysozyme produced is no longer temperature sensi ti ve. The crystal structure of P22 lysozyme is not yet solved. Assuming that the structures of T4 lysozyme and P22 lysozyme are similar , one can examine the positions of equivalent residues in the T4 lysozyme structure. The spatial arrangement of the residues changed by the secondary site mutations and the original substitution can then be visualized. The mutations discussed above all map far from the original mutation on the T4 three dimensional model. A substitution of leucine for tyrosine at position 22 is compensated for by the double mutation of arg18ser and ser23lys . When the equivalent residues are mapped on the T4 three dimensional model the changes map in close proximity to the