9513429 Baldwin An understanding of the processes of protein folding is crucial to full realization of the potential of modern biotechnology. In spite of intense efforts over the past ca. 30 years, many questions remain. Of central concern today is the role of chaperones in the folding process. Current dogma holds that the native structure of a protein is the most stable structure possible, but the existence of chaperones raises the possibility that chaperones might allow proteins to assume conformations that do not form fast enough in their absence to be of biological significance. We propose to use bacterial luciferase to probe the roles of chaperones in determining the rates of formation of native and nonnative structures. Luciferase is an excellent model system for such studies for numerous reasons, including the ease and sensitivity of the assay (the enzyme emits light) and the dimeric structure of the enzyme. Since the enzyme is composed of two nonidentical subunits, ( and (, it is possible to investigate the folding of each subunit in isolation, and to investigate the assembly of the enzyme in the absence of more complicating folding processes. We expect that, through these studies, we will be able to ascertain the extent to which the chaperones interact with and guide the folding of thepolypeptide chain during biosynthesis on the ribosome. %%% The objective of this project is to understand the biosynthetic folding pathway of the ( subunit of bacterial luciferase and the role(s) played by molecular chaperones in this process. "Biosynthetic folding" refers to the processes associated with folding during synthesis of the polypeptide on a ribosome. The cell free protein synthesis strategies serve as a model for protein synthesis and folding within the living cell. Bacterial luciferase is a cytoplasmic heterodimer capable of unassisted spontaneous refolding from the denatured state. It has been shown that the ( subunits folds cotranslationally, contributing to the fast acqui sition of native structure. Structural features of the ribosome-bound nascent ( subunit will be probed by limited proteolysis. A fragment of the ( subunit corresponding to the nascent polypeptide immediately prior to release, i.e., without the C-terminal region which is sheltered by the ribosome, will be isolated and probed by limited proteolysis, circular dichroism and fluorescence. This large fragment and a fragment representing the C-terminal portion of ( will be used to analyze the kinetics of folding and assembly of bacterial luciferase. These experiments will permit development of a more complete model of biosynthetic folding/assembly with purified components. The nature of the ( subunit species capable of association with the ( subunit following release from the ribosome will be analyzed and compared with the heterodimerization-competent ( species formed during refolding, to understand apparent differences in the rates of formation of luciferase during biosynthesis of ( and refolding of (. It has been shown that ( subunit can interact with chaperones, and that certain chaperones are able to accelerate formation of enzymatically active heterodimer. The mechanism of this phenomenon and its relation to biosynthetic folding will be studied. During refolding of the pure protein, the ( subunit acquires alternative conformations which cannot interact with (. The effect of chaperones on the distribution of ( subunit between these alternative conformations will be examined. I
