Surface plasmon resonance analysis of the mechanism of binding of apoA-I to high density lipoprotein particles

Abstract

The partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between HDL-bound and-unbound states is an integral part of HDL metabolism. We used the surface plasmon resonance (SPR) technique to monitor in real time the reversible binding of apoA-I to HDL. Biotinylated human HDL2 and HDL3 were immobilized on a streptavidin-coated SPR sensor chip, and apoA-I solutions at different concentrations were fl owed across the surface. The wild-type (WT) human and mouse apoA-I/HDL interaction involves a two-step process; apoA-I initially binds to HDL with fast association and dissociation rates, followed by a step exhibiting slower kinetics. The isolated N-terminal helix bundle domains of human and mouse apoA-I also exhibit a two-step binding process, consistent with the second slower step involving opening of the helix bundle domain. The results of fl uorescence experiments with pyrene-labeled apoA-I are consistent with the N-terminal helix bundle domain interacting with proteins resident on the HDL particle surface. Dissociation constants (K d) measured for WT human apoA-I interactions with HDL2 and HDL3 are about 10 μM, indicating that the binding is low affinity. This Kd value does not apply to all of the apoA-I molecules on the HDL particle but only to a relatively small, labile pool. Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.