Basis of substrate binding by the chaperonin GroEL.

The molecular chaperonins are essential proteins involved in protein folding, complex assembly, and polypeptide translocation. While there is abundant structural information about the machinery and the mechanistic details of its action are well studied, it is yet unresolved how chaperonins recognize a large number of structurally unrelated polypeptides in their unfolded or partially folded forms. To determine the nature of chaperonin-substrate recognition, we have characterized by NMR methods the interactions of GroEL with synthetic peptides that mimic segments of unfolded proteins. In previous work, we found using transferred nuclear Overhauser effect (trNOE) analysis that two 13 amino acid peptides bound GroEL in an amphipathic alpha-helical conformation. By extending the study to a variety of peptides with differing sequence motifs, we have observed that peptides can adopt conformations other than alpha-helix when bound to GroEL. Furthermore, peptides of the same composition exhibited significantly different affinities for GroEL as manifested by the magnitude of trNOEs. Binding to GroEL correlates well with the ability of the peptide to cluster hydrophobic residues on one face of the peptide, as determined by the retention time on reversed-phase (RP) HPLC. We conclude that the molecular basis of GroEL-substrate recognition is the presentation of a hydrophobic surface by an incompletely folded polypeptide and that many backbone conformations can be accommodated.