The projects proposed address two areas where fundamental studies are critically needed: the mechanism of folding of predominantly beta-sheet proteins, and the mechanism of action of the Hsp70 family of molecular chaperones.
The specific aims that will be pursued are: 1) Based on the model for folding emerging from studies in the previous grant period, we will use cellular retinoic acid binding protein I (CRABP I), a predominantly beta- sheet protein with an ill-defined hydrophobic core, to develop principles about the relation between amino acid sequence and the folding of beta sheet proteins. We seek to understand how hydrophobic interactions influence CRABP I folding, how important local sequence conformational preferences are in CRABP I folding, what residue interactions specify the topology of CRABP I, and what the nature of intermediate states is for this beta-rich protein. To address these aims, we will carry out sequence and structure analysis of CRABP I and its family members; perform protein engineering experiments to dissect the contributions of specific residues and groups of residues in folding; assess the influence of local sequences by study of peptide fragments and by kinetic analysis of sub-millisecond folding events; and explore the dynamics of CRABP I using experimental and computational methods under conditions where the protein is stable and under unfolding conditions. 2) We will carry out structure-function studies of two highly homologous members of the Hsp70 family of moleuclar chaperones, DnaK, the E. coli representative of this family, and BiP, the mammalian representative that resides in the lumen of the endoplasmic reticulum. We seek to understand how ATP binding to the N-terminal domain of Hsp70s leads to reduced affinity of substrate binding to the C-terminal domain, and how two highly homologous Hsp70s have the common ability to recognize unfolded substrates, yet show differential sequence preferences in their substrates. To address these aims, we will analyze in detail mutants that have impaired allosteric communication, using NMR structure determination and biochemical assays. We will compare structures with and without substrate bound. We will incorporate tryptophan and cystein residues in strategic locations, attach fluorophores to the Cys residues, and measure interdomain movements by fluorescence energy transfer-based distance. Our studies of the folding of a predominantly beta- sheet protein will contribute to an improved understanding of the origin of amyloidogenic diseases, such as Alzheimer's, bovine spongiform encephalomyelitis, and Huntington's, which appear to arise from misfolding events. Elucidation of the mechanism of action of the Hsp70 family of molecular chaperones will enhance our understanding of how organisms respond to heat shock and stress, as well as the origins of diseases postulated to arise from defects in Hsp70-facilitated protein folding, such as cystic fibrosis and p53-related cancers.
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