The objective of this proposal is to understand the molecular mechanism by which chaperoning ring-complexes mediate protein folding in ATP- dependent reactions. The 'chaperonins' (cpn60s; Hsp60 family of molecular chaperones) in bacteria, mitochondria and chloroplasts form large cylinders composed of two stacked heptameric rings of 60 kDa subunits. They are functionally regulated by a co-chaperoning (cpn 10) a heptameric ring of 10 kDa subunits. The recent discovery of the chaperoning-related TCP-1 ring-complex (TRiC) in the eukaryotic cytosol underscores the general importance of this type of molecular chaperone for cellular protein folding. By reconstituting the function of E. coli cpn60/cpn10 in vitro, we have established that the basic role of the chaperonins is to support polypeptide chain folding rather than oligomeric assembly of protein subunits. We have now discovered by electron microscopic image analysis that the chaperoning cylinder accommodates the substrate protein within its central cavity presumably in the conformation of a compact folding intermediate. Building on this foundation, we plan to perform a detailed structural and functional analysis of the cpn60/cpn10 system and of TRiC. We will test our hypothesis that cellular protein folding proceeds within a protected environment provided by the internal space of the chaperoning ring- complex. The following specific aims will be addressed in a biochemical and biophysical approach: I. Define the binding sites on cpn60 for the regulatory co-chaperoning cpn10 and for nucleotides by crosslinking followed by peptide analysis using reversed-phase HPLC, mass spectrometry and sequencing. The significance of the results obtained by this approach will be tested by site-specific mutagenesis of cpn60. II. Analyze the structural basis for the interaction of chaperonins with unfolded substrate protein. Sequences that bind specifically to the chaperoning will be identified by limited proteolysis of bound protein and analysis of the resulting fragments. The binding site(s) for substrate protein will be mapped within the domain-structure of the chaperoning. The conformation of chaperone-bound model proteins will be analyzed by the hydrogen-exchange techniques coupled with mass spectrometry and nuclear magnetic resonance spectroscopy. III. Determine how ATP-dependent release from the chaperoning leads to productive folding. Chaperoning-mediated folding will be dissected into intermediate steps by defining the interactions between the participating components throughout the process. Large proteases and site-specific antibodies against substrate protein will be used to analyze how far folding progresses on cpn60. Oriented immobilization of cpn60 on agarose beads or lipid monolayers will serve to test the functional significance of the asymmetry in the cpn60-cpn10 complex. IV. Analyze the structure of chaperoning complexes with bound substrate by three-dimensional electron microscopic reconstruction. Domains of the chaperoning subunits will be defined by limited proteolysis and their topology within the chaperoning complex will be determined by electron microscopy using domain-specific antibodies. The crystallization of the cpn60 oligomer or of its stably folded domains will be attempted.
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