MECHANISM OF CHAPERONE MEDIATED PROTEIN FOLDING

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The long term goal of our research is to understand how efficient protein folding is achieved in the complex intracellular milieu. We are particularly interested in understanding how molecular chaperones alter the nature of protein folding reactions, and how the cocktail of molecular chaperones helps determine the spectrum of proteins that can fold in a particular organism. Knowledge of how efficient folding is accomplished in vivo could have important practical benefits. For example, such studies could lead to a better understanding of the molecular basis of a wide variety of diseases involving either misfolding or the accumulation of aggregated proteins such as Cystic Fibrosis, alpha1-Antitrypsin Deficiency and Prion based encephalopathies, as well as allow for the more efficient production of proteins for research and commercial purposes. The present studies will focus on the so called "chaperonin" family of ring-shaped molecular chaperones. The chaperonins form a ubiquitous and abundant family of proteins whose members play an essential role in assisting folding in both prokaryotes and eukaryotes. Recent studies provide a wealth of data on the structure and reaction cycle of the E. coli chaperonin GroEL and its co-chaperonin GroES. These studies establish that GroEL mediated folding proceeds by multiple round of binding and release of non-native polypeptides, and that protein folding is generally initiated and potentially completed while a protein remains sequestered within the GroEL central cavity under GroES. Taking advantage of the ability to dissect a GroEL-GroES mediated protein folding reaction into specific well- defined steps, we will investigate how GroEL uses the energy of ATP hydrolysis to assist protein folding. We will also use various biochemical and biophysical approaches, including solution binding studies and X-ray crystallography, to characterize how GroEL recognizes unfolded proteins. Our specific aims are to: (1) Investigate the effect of sequestering a polypeptide within a GroEL-GroES complexes on its folding pathway. (2) Examine the mechanism and functional significance of cycles of polypeptide release and rebinding during a chaperonin-mediated folding reaction. (3) Biochemically characterize how GroEL recognizes unfolded substrates. (4) Examine the structural basis of polypeptide recognition by GroEL.