FOLDING OF PHAGE P22 STRUCTURAL PROTEINS

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Within cells, newly synthesized polypeptides fold within a complex milieu of DNA, RNA and extremely high concentrations of proteins. At these high protein concentrations the folding of polypeptides in vitro fails, resulting in aggregation rather than productive folding. Yet, proteins are able to fold in vivo. This paradox has recently been resolved by the discovery of a class of proteins known as molecular chaperones. Molecular chaperones function by facilitating the folding of a subset of polypeptides by interacting with their folding intermediates to keep them from aggregating. Clearly, molecular chaperones differentiate among folding intermediates since only a subset require assistance for productive folding. However, what distinguishes a recognized folding intermediate from a non-recognized one is an enigma. The long term goal of this project is to understand the features of folding intermediates that are recognized by chaperones and how chaperones facilitate the folding of substrate polypeptides in vivo. Coat protein from the Salmonella bacteriophage P22 provides a unique model system with which to study the features of in vivo folding intermediates that are recognized by the molecular chaperones, GroEL and GroES; coat protein mutants, whose folding is defective at high temperature, require GroEL and GroES for productive folding and assembly. Consequently, these single amino acid substitutions perturb the folding of the mutant coat polypeptides in such a way that they become substrates of GroEL and GroES. An additional strength of this model system is this ability to easily select for mutants affected in folding. In this proposal biophysics, biochemistry, and genetics will be used to probe the interactions of GroEL and GroES with coat polypeptide substrates in vivo and in vitro by correlating results obtained from in vivo folding experiments with those obtained from kinetic studies in vitro. The effect of the amino acid substitutions on the kinetics folding of coat protein will be analyzed in vitro using intrinsic and extrinsic fluorescence, and circular dichroism. The binding pocket of GroEL will be probed by fluorescence quenching and accessibility of substrate polypeptides to protease. Conditions for the rescue of proper folding of the mutant coat proteins by GroEL and GroES in vitro will be determined. The ability of GroEL and GroES to correct folding in vivo will be ascertained at normal, increased, or decreased levels of GroEL and GroES. GroEL and GroES whose function has been altered by mutation will be tested for the ability to properly fold substrate polypeptides in vivo. Moreover, a genetic approach will be used to investigate the interactions that occur during folding, both within a folding intermediate and between folding intermediates and GroEL, by isolating intragenic second site suppressors of the defective coat proteins in cells producing wild-type or defective GroEL. The variety of approaches proposed will allow singular insight in the mechanism of protein folding in vivo. This insight is important since many serious diseases, such as Alzheimer's disease, are caused by the misfolding of proteins.