Surface plasmon resonance analysis of the mechanism of binding of apoA-I to high density lipoprotein particles.
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 HDL(2) and HDL(3) were immobilized on a streptavidin-coated SPR sensor chip, and apoA-I solutions at different concentrations were flowed 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 fluorescence 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 HDL(2) and HDL(3) are about 10 microM, indicating that the binding is low affinity. This K(d) value does not apply to all of the apoA-I molecules on the HDL particle but only to a relatively small, labile pool.
Lipid Research Group, Division of Gastroenterology, Hepatology, and Nutrition, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4318, USA.
Sissel
S
Lund-Katz
David
D
Nguyen
Padmaja
P
Dhanasekaran
Momoe
M
Kono
Margaret
M
Nickel
Hiroyuki
H
Saito
Michael C
MC
Phillips
D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance
D016632:Apolipoprotein A-I; D055767:Immobilized Proteins; D008075:Lipoproteins, HDL