Protein folding in vivo is now understood to be assisted by a special class of helper proteins collectively known as chaperones. The work proposed in this grant will concentrate on the family of 70 kDa heat shock proteins (Hsp70), which is conserved from bacteria to mammals. The proteins are involved in the earliest stages of peptide translation and bind to nascent peptide chains and peptide chains destined for transport to cellular organelles. In addition, these proteins bind to proteins damaged by thermal and other stress (heat shock) or mutation. In this grant application, we propose to determine the three-dimensional structures and the dynamics of the peptide binding domains of two eukaryotic Hsp 70 proteins, the Hsc found in the cytosol and the BiP protein of the endoplasmic reticulum. In addition, the structures of complexes of these proteins with small peptides will be studied. Multi- nuclear, multi-dimensional NMR methods will be used; the information will be correlated with function using site-directed mutagenesis in a collaborative fashion. The objective of these studies is to gain insight in the basis of the function of these chaperone proteins. With this information, we may infer and rationalize the apparent specificity of the chaperones for hydrophobic peptides. We will also gain insight in possible conformational changes that modulate the affinity of Hsp70s for their targets. The structures of the bound peptides will indicate their mode of binding and will help resolve the question whether the Hsp70s are merely solubilizing unfolded proteins or if initial stages of protein folding are taking place in their binding cleft. The dynamic information will be integral part of the interpretation processes. Protein folding is very basic to cell functioning; it is only with properly and timely folding proteins that the cell can perform its complicated regulatory functions and growth cycles. The insight in the structural basis of in vivo protein folding that will be gained from the proposed studies is therefore of relevance to the understanding of these growth cycles and their disturbances.
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