Use of site-directed mutagenesis to obtain isomorphous heavy-atom derivatives for protein crystallography: cysteine-containing mutants of phage T4 lysozyme.
Five different cysteine-containing mutants of the lysozyme from bacteriophage T4 were used to explore the feasibility of using site-directed mutagenesis to generate isomorphous heavy-atom derivatives for protein crystallography. Cysteines 54 and 97, present in wild-type lysozyme, can be readily reacted with mercuric ion to produce an excellent isomorphous heavy-atom derivative. Mutants with an additional cysteine at position 86, 146, 153 or 157, or with Cys 97 replaced by Val, were engineered by site-directed mutagenesis. The mutant lysozyme Thr 157----Cys reacts with mercuric chloride to give an excellent new derivative although Cys 157 is only approximately 60% substituted with the heavy atom. The cysteine at position 146 is largely buried but reacts readily with mercuric chloride. In this case the isomorphism is poor and the resultant derivative is of marginal quality. Cys 153 reacts rapidly with mercuric ion but the derivative crystals do not diffract. The mutant Pro 86----Cys does not yield a particularly good heavy-atom derivative. This is due in part to a loss of isomorphism associated with the mutation. In addition, Cys 86 shows very little reactivity towards mercurials even though it is fully exposed to solvent. The mutation Cys 97----Val was used to explore the possibility of creating an independent derivative by deleting a heavy-atom site already present in wild-type lysozyme. In all cases that were tested, the quality of the heavy-atom derivative was improved by using as an isomorphous pair mercury-substituted mutant versus non-substituted mutant rather than mercury-substituted mutant versus (non-substituted) wild-type lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine; D008628:Mercury; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009113:Muramidase / Q000235:genetics*; D009154:Mutation; D015202:Protein Engineering; D013604:T-Phages / Q000201:enzymology / Q000235:genetics; D014961:X-Ray Diffraction
D009113:Muramidase; D008628:Mercury; D003545:Cysteine