FOLDING OF PHAGE P22 STRUCTURAL PROTEINS

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Within cells, newly synthesized polypeptides fold in a complex environment of high concentrations of macromolecules. Thus, in vivo proteins are able to fold under conditions that would cause in vitro folding to fail. In order to prevent aggregation, molecular chaperones facilitate the folding of certain proteins by interacting with unfolding intermediates or partially folded forms. Molecular chaperones appear to be able to differentiate among folding intermediates in order to identify the subset required for productive folding. The factors that distinguish a substrate for a chaperone from a non-recognized polypeptide are still unknown. Coat protein of the P22 phage is an appropriate system for investigating this question. A strength of this system is the ability to select for mutants that require chaperones to fold. The wild type protein does not require the chaperones GroEL or GroEs to fold but point mutants have been characterized which require these chaperones for folding. Global suppressor mutants that rescue these mutants have also been discovered. In addition, the folding of coat protein can be decoupled from its assembly, thereby allowing the separate investigation of the control exterted by the primary amino acid sequence on these processes. In this proposal, a combination of biochemical, biophysical and genetic approaches will be used to study the in vivo and the in vitro folding and assembly of the coat protein of phage P22. The comparison of quantitative in vitro experiments with the results of in vivo measurements will aid in the elucidation of how the primary amino acid sequence directs folding and assembly. These experiments will also provide insight into interactions with GroEL and GroES that are required for folding. The goals of the research are: 1) to determine the role of crucial amino acids in the folding and assembly of coat protein; 2) investigate the interaction of substrate polypeptides with GroES and GroEL in vitro and in vivo and; 3) examine the effects of mutations at the subunit interface on folding and assembly. The range of approaches will allow insight into the factors which control protein folding and assembly in vivo. This insight is important since misfolding and/or protein misassembly has been implicated in serious diseases.