How a newly translated linear polypeptide folds into a functional native protein is the central issue in protein biochemistry today. The purpose of the proposed research is to delineate the mechanism by which the subunits of bacterial luciferase fold and assemble into the functional heterodimeric enzyme in vivo. Genes for the two subunits, luxA and luxB, have been cloned and expressed in E. coli. Thus, the subunits can be easily prepared. Moreover, their structures can be readily altered by oligonucleotide-directed site specific mutagenesis. The ease with which low light intensities can be measured permits the detection of specific activites in mutants over a range of six orders of magnitude. Thus, the luciferase heterodimer provides an ideal system for studying the principles which govern protein folding in vivo. In this project mutants of the subunits will be prepared, and the way they combine to form active enzyme will be analyzed through kinetic and physical characterization of the folding and assembly process.
