Cholesteryl ester diffusion, location and self-association constraints determine CETP activity with discoidal HDL: Excimer probe study

•Cholesteryl pyrene decanoate excimer in discoidal HDL follows sphere-of-action model.•CETP-catalyzed CPD transfer from donor to acceptor HDL is kinetically heterogeneous.•The fast exchanging CPD pool is much larger in HDL with POPC compared to that in DPPC.•Acceptor particles noncompetitively inhibit probe transfer.•CPD diffusion, location and association underlie CETP activity with discoidal HDL. The transfer of cholesteryl ester by recombinant cholesteryl ester transfer protein (CETP) between reconstituted discoidal high-density lipoprotein (rHDL) was studied. Particles contained apolipoprotein A-I, unsaturated POPC or saturated DPPC and cholesteryl ester as cholesteryl 1-pyrenedecanoate (CPD) or cholesteryl laurate (CL) in donor and acceptor rHDL, respectively. Probe dynamics fulfilled the quenching sphere-of-action model. The cholesteryl ester exchange between donor and acceptor particles was characterized by a heterogeneous kinetics; the fast exchanging CPD pool was much higher in a case of POPC compared to DPPC complexes. Probe fraction accessible to CETP increased with temperature, suggesting a more homogeneous probe distribution. Noncompetitive inhibition of probe transfer by acceptor particles was observed. The values of Vmax (0.063μMmin−1) and catalytic rate constant kcat (0.42s−1) together with a similarity of Km (0.9μM CPD) and KI (2.8μM CL) values for POPC-containing rHDL suggest the efficient cholesteryl ester transfer between nascent HDL with unsaturated phosphatidylcholine in vivo. The phospholipid matrix in discoidal HDL may underlie CETP activity through the self-association, diffusivity and location of cholesteryl ester in the bilayer, the accessibility of cholesteryl ester to cholesterol-binding site in apoA-I structure and the binding of cholesteryl ester, positionable by apoA-I, to CETP.