360-MHz nuclear magnetic resonance and laser photochemically induced dynamic nuclear polarization studies of bile salt interaction with porcine colipase A.

Abstract

Porcine pancreatic colipase possesses a particular three‐dimensional surface domain containing six out of the seven aromatic residues of the molecule in a highly hydrophobic environment [P. Canioni and P. Cozzone (1979) Biochimie (Paris) 61, 343–354; J. Wieloch, B. Borgström, K. E. Falk and S. Forsen (1979) Biochemistry, 18, 1622–1628]. The domain corresponds to the 49–57 and 77–86 β‐sheet fragments brought into spatial proximity by protein folding [P. Canioni, P. Cozzone and L. Sarda (1980) Biochim. Biophys. Acta, 621, 29–42]. The identification of this specific domain as being the lipid binding site on colipase is proposed on the basis of the NMR and photochemically induced dynamic nuclear polarization (photo‐CIDNP) studies of the complexes of colipase with organized bile salt micelles.The binding of taurodeoxycholate micelles specifically perturbs the proton NMR ring resonances of Tyr‐I and Tyr‐II (Tyr‐56 and Tyr‐57) and His‐II (His‐86) together with several aliphatic resonances, reflecting the involvement of the hydrophobic aromatic domain in micelle fixation. In the presence of colipase, specific shifts and broadening of the methyl groups at position 18 and 21 of the sterane ring of taurodeoxycholate are observed and suggest that the hydrophobic side of the bile salt is primarily involved. The strong photo‐CIDNP effects of Tyr‐I and Tyr‐II in the presence of lumiflavin dye, which have been described on free colipase [P. Canioni, P. Cozzone and R. Kaptein (1980) FFBS Lett. 111, 219–222] are totally suppressed in the presence of taurodeoxycholate or chenocholamine micelles, indicating that due to the protection by the bile salt, the aromatic surface residues are no longer accessible. The pH dependence of the NMR perturbations and photo‐CIDNP effects observed in the colipase‐micelle complexes confirms that (1) the hydrophobic aromatic domain is directly involved and (2) the driving force of the primary micelle binding is essentially hydrophobic.A general model for micelle binding to colipase is proposed and involves a two‐step mechanism. Initially, the lipid hydrophobic binding site participates in the building of the bound taurodeoxycholate aggregate to form a mixed micelle through hydrophobic surface interactions (stoichiometric complex). At higher bile salt concentrations, polar forces might account for further growth of the micellar structure bound on colipase.