GroEL is an essential Escherichia coli molecular chaperon that uses ATP to facilitate correct folding of a range of proteins in a cell. Central to the GroEL substrate diversity is how GroEL recognizes the substrates. The interaction between GroEL and substrate has been proposed to be largely hydrophobic because GroEL interacts with proteins in non-native conformations but not in native forms. Analysis of GroEL substrate proteins reveals that one of its main substrates are proteins with alphabeta folding domains, suggesting that GroEL may stabilize the collapsed alphabeta core by binding to hydrophobic surfaces that are usually buried between the alpha and beta elements. In this study, we characterize the interaction between GroEL and a peptide derived from our previous selection via a phage display method. NMR studies map the peptide-binding site to the region containing Helices H and I, which is consistent with evidence that this region comprises the primary substrate-binding site. The peptide is largely unstructured in solution but adopts a helical conformation when bound to the GroEL apical domain with a moderate affinity (K(d) = 17.1 +/- 2.5 microm). The helical conformation aligns residues to form an amphipathic structure, and the hydrophobic side of this amphipathic helix interacts with GroEL as suggested by fluorescence quenching studies. Together with previous structural studies on the GroEL-peptide complexes, our work supports the notion that the amphipathic secondary elements in the substrate proteins may be the structural motif recognized by GroEL.