Structural Requirements for Protein Function Studied by in Vitro Mutagenesis on Beta-Lactamase

Citation

Dalbadie-McFarland, Gloria (1985) Structural Requirements for Protein Function Studied by in Vitro Mutagenesis on Beta-Lactamase. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/jhx0-q907. https://resolver.caltech.edu/CaltechTHESIS:04052019-162706704

Abstract

The study of naturally occurring variants of proteins has been successfully used for a long time to assign roles to structural elements in a protein and to correlate functional requirements with the nature of these structural elements.

Advances in techniques for DNA synthesis and DNA sequencing, along with the development of recombinant DNA techniques now allow one to clone the gene for a protein and then to modify it at will. In this way, presumably any and all aminoacid substitutions can be engineered into any protein whose gene has been cloned, characterized and expressed.

Beta-lactamase has been used as a model system in which to study the feasibility of the approach, and it has been demonstrated that not only is it possible to introduce specific predetermined changes in the structure of a protein, but that these mutants can in turn serve as substrates for further modifications. An inactive enzyme can be used to search for a broad range of structural requirements by imposing selective conditions that require a function for the survival of the host organism for the mutant protein.

Four variants of beta lactamase, one of which is catalytically active, have been obtained by site specific mutagenesis. In the inactive mutants the conserved active site sequence -ser70-thr71- was altred to either -thr70-ser71-, -thr70-thr-71- or -arg-70-thr71-; a variant in which a disulfide bond was removed by mutating one of the only two cysteines in E. coli beta-lactamse to serine was found to be active. In addition, a revertant to activity has been obtained from one of the the inactive mutants (-thr70-ser71-). The revertant is different in aminoacid sequence (-ser70-ser71-) and in some of its properties from the wild type enzyme while still having catalytic activity. No revertants with aminoacid substitutions at a secondary site were found.

Both the catalytically active revertant and the mutant lacking the disulfide bridge were found to have reduced thermal stability. The rate of secretion of three of these mutants (ser70→thr, thr71→ser and ser70→thr/thr71→ser) was compared to that of the wild type beta lactamase and no significant differences were found. The -arg70-ser71- mutant will be used to identify chemical mutagens and carcinogens that induce GC to TA transversions by collaborators P. L. Foster and D. Botstein.

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