Biochemical and structural studies of Escherichia coli chaperone groel-substrate interaction

The E. coli molecular chaperone GroEL and its co-chaperone GroES function together as essential cellular machinery which assists protein folding in an ATP-dependent manner. The double-ring GroEL tetradecamer encapsulates non-native substrate proteins into the central cavity and promotes their folding. The current view is that GroEL assists protein folding by isolating the substrate protein from other cellular molecules in an effectively infinite dilution, and unfolding the off-pathway intermediates to free the misfolded substrate from kinetic traps.;One of the most intriguing aspects of GroEL function is that GroEL recognizes a variety of substrates with different structures and functions. To unravel the mechanism of GroEL-substrate interaction, we adopted a reductionist approach to study the interaction between the GroEL apical domain, the substrate binding site of GroEL, and small peptides, using a combination of biochemical and structural methods. To validate this approach, we showed that a peptide SBP, previously identified to bind to the GroEL substrate binding site, was able to compete with GroEL substrate proteins in the binding to GroEL. We used various NMR techniques to characterize several peptides in their interactions with the GroEL apical domain. One of the peptides was found to interact with the region containing Helix H and I on the apical domain and adopted a helical conformation in the bound form. The helical conformation aligns the residues to form an amphipathic structure, and the fluorescence studies suggested that the hydrophobic side interact with GroEL. Combining with previous structural studies, our results here support that the role of GroEL may be involved in preserving and stabilizing the amphipathic secondary structures of substrate proteins. Due to various technical difficulties, structural studies of the other peptides with GroEL apical domain were incomplete. Nevertheless, these studies suggest that the peptides bind to the region of Helix H and I, and mainly adopt helical conformations. The work presented in the thesis enables a more detailed understanding of GroEL-substrate recognition mechanism.