Molecular chaperones are involved in a wide range of essential cellular processes: protein synthesis, molecular assembly, translocation, degradation, and folding. The E. coli molecular chaperone GroEL, along with its co-chaperone GroES, increases the efficiency of protein folding in vivo, using an ATP-driven mechanism. In the GroEL-facilitated folding process, first GroEL sequesters the aggregation-prone nonnative forms of proteins from the complex cellular environment within its central cavity. Then with the actions of ATP binding/hydrolysis and GroES binding, the protein is allowed to carry out its initial folding events within an isolated """"""""folding chamber"""""""" formed by GroEL/GroES. The long-term goals of this proposal are to elucidate the structural features of the interaction of GroEL and substrate proteins, to understand the mechanism of GroEL-facilitated protein folding in a structural context, and to further structural knowledge of molecular chaperones function in general. Various biochemical and biophysical techniques, including phage display, fluorescence spectroscopy/polarization and X-ray crystallography, will be used to determine how GroEL recognizes the substrate proteins, and to understand the energetics of ATP binding/hydrolysis.
Three specific aims are to: 1) Select for small peptides that interact with the substrate binding domain of GroEL using a phage display method. 2) Study the interplay of GroEL-substrate by determining the structures of the substrate-trapped GroEL assemblies using X-ray protein crystallography and NMR, and carry out structure-guided mutational studies. 3) Create model polypeptide substrates for GroEL based on the peptides selected in 1) and use them to study the functional role of nucleotide binding/hydrolysis and the mechanism of GroEL-assisted protein folding. Knowledge of protein folding and the role of molecular chaperones in facilitating the folding process will contribute to a better understanding of folding-related human diseases, such as Cystic Fibrosis, Alzheimer's, Prion diseases and cataracts, at the molecular level, and could lead to the design of novel therapeutic approaches.
