Electrostatic as well as hydrophobic interactions are important for the association of Cpn60 (groEL) with peptides

The interactions of groEL with five N-dansyl peptides were investigated by means of a fluorescence binding assay. The peptides studied (Ba-mph, B-hphil, A(amph), A(hphil), N-amph) were designed and synthesised as systematic variants of each other in terms of their patterns of charge and hydrophobicity. Fluorescence data were analysed using a fluorescence modified, y-reciprocal linearised form of the Benesi-Hildebrand equation which was derived from first principles and verified by theoretical simulations. Under optimal conditions, apparent dissociation constants, K-d, were obtained in the mu M range. At physiologically relevant ionic strengths, only two peptides (basic amphiphilic B-amph and neutral amphiphilic N-amph) interacted with groEL whilst a third peptide (acidic amphiphilic A(amph)) was able to interact but only at very high ionic strength (> 1 mol kg(-1)). Thermodynamic (van't Hoff) analysis of the tightest binder, basic amphiphilic B-amph peptide, revealed endothermic binding and a large positive entropy, Delta S-bind(0), consistent with a mixed binding mode involving both hydrophobic and electrostatic interactions. At physiologically relevant ionic strengths, positively charged amino acid residues appear to augment hydrophobic binding interactions with groEL by electrostatic attraction whilst negatively charged amino acid residues oppose short-range hydrophobic interactions with electrostatic repulsion. In conclusion, whilst a principal means of interaction between groEL and a peptide or partially folded protein substrate is certainly hydrophobic, electrostatic effects can modulate or even overwhelm this interaction.