Basis of substrate binding by the chaperonin GroEL

The molecular chaperonins are essential proteins involved in protein foldin g, 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 re cognition, we have characterized by NMR methods the interactions of GroEL w ith synthetic peptides that mimic segments of unfolded proteins. In previou s work, we found using transferred nuclear Overhauser effect (trNOE) analys is that two 13 amino acid peptides bound GroEL in an amphipathic a-helical conformation. By extending the study to a variety of peptides with differin g sequence motifs, we have observed that peptides can adopt conformations o ther than or-helix when bound to GroEL. Furthermore, peptides of the same c omposition exhibited significantly different affinities for GroEL as manife sted 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 p eptide, as determined by the retention time on reversed-phase (RP) HPLC, We conclude that the molecular basis of GroEL-substrate recognition is the pr esentation of a hydrophobic surface by an incompletely folded polypeptide a nd that many backbone conformations can be accommodated.