Local Perturbations by Ligand Binding of Hydrogen Deuterium Exchange Kinetics in a Four-helix Bundle Protein, Acyl Coenzyme A Binding Protein (ACBP)

Abstract

Amide hydrogen exchange kinetics of the individual amides in a four-helix bundle protein, acyl-coenzyme A binding protein, have been studied by nuclear magnetic resonance spectroscopy. The kinetics of amides with exchange rate constants in the range of 10 2.5to 10 6.5S 1at pH 6.65 in free protein and the ligand-protein complex have been measured, and the effect of binding the ligand, palmitoyl-coenzyme A, on individual exchange rates has been analysed. Specific correlations between exchange kinetics and the structural properties of the individual amides known from the three-dimensional structure of acyl-coenzyme A binding protein have been examined. Furthermore, an analysis has been performed comparing the structural perturbations of the protein-ligand interactions known from the three dimensional structure of the complex of palmitoyl-coenzyme A and acyl-coenzyme A binding protein with the ligand-induced perturbations on the amides exchange kinetics. Finally, the ligand-induced perturbations on hydrogen exchange have been compared with those on 15N relaxation. The results suggest that hydrogen exchange kinetics in the individual sites of acyl-coenzyme A binding protein are primarily determined by local structural features; they show that ligand binding gives rise mainly to changes localized at the sites of interaction between protein and ligand; they imply that the perturbation of exchange kinetics caused by ligation can be either, as in one example a local stabilisation of the pre-exchange equilibrium induced by formation of a hydrogen bond, or as seen here in several examples a reduction of the dynamic processes that lead to the opening and closing processes of the pre-exchange equilibrium. The results seem not to indicate changes in the rate of the final chemical exchange step. f2 f2 Abbreviations used: ACBP, acyl coenzyme A binding protein; CoA, coenzyme A; C12, chymotrypsin inhibitor 2 from barley; ESI-MS, electrospray ionization mass spectrometry; GuHCl, guanidine hydrochloride; HSQC, heteronuclear single quantum coherence; n.m.r., nuclear magnetic resonance; p.p.m., parts per million; TPPI, time proportional phase incrementation.