Enthalpy-Driven Apolipoprotein A-I and Lipid Bilayer Interaction Indicating Protein Penetration upon Lipid Binding † Cristina Arnulphi,* ,‡ Lihua Jin, § M. Alejandra Tricerri, ‡ and Ana Jonas ‡ Department of Biochemistry, UniVersity of Illinois at Urbana-Champaign, and Department of Chemistry, DePaul UniVersity, Chicago, Illinois 60614 ReceiVed NoVember 25, 2003; ReVised Manuscript ReceiVed March 9, 2004 ABSTRACT: The interaction of lipid-free apolipoprotein A-I (apoA-I) with small unilamellar vesicles (SUVs) of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with and without free cholesterol (FC) was studied by isothermal titration calorimetry and circular dichroism spectroscopy. Parameters reported are the affinity constant (K a ), the number of protein molecules bound per vesicle (n), enthalpy change (ΔH°), entropy change (∆S°), and the heat capacity change (∆C p °). The binding process of apoA-I to SUVs of POPC plus 0-20% (mole) FC was exothermic between 15 and 37 °C studied, accompanied by a small negative entropy change, making enthalpy the main driving force of the interaction. The presence of cholesterol in the vesicles increased the binding affinity and the R-helix content of apoA-I but lowered the number of apoA-I bound per vesicle and the enthalpy and entropy changes per bound apoA-I. Binding affinity and stoichiometry were essentially invariant of temperature for binding to SUVs of POPC/FC at a molar ratio of 6/1 at (2.8-4) × 10 6 M -1 and 2.4 apoA-I molecules bound per vesicle or 1.4 × 10 2 phospholipids per bound apoA-I. A plot of ∆H° against temperature displayed a linear behavior, from which the ∆C p ° per mole of bound apoA-I was calculated to be -2.73 kcal/(mol‚K). These results suggested that binding of apoA-I to POPC vesicles is characterized by nonclassical hydrophobic interactions, with R-helix formation as the main driving force for the binding to cholesterol-containing vesicles. In addition, comparison to literature data on peptides suggested a cooperativity of the helices in apoA-I in lipid interaction. Protein and lipid interactions are known to define structure and function of apolipoproteins and thus modulate the metabolism of lipoproteins (1). One class of lipoproteins, the human high-density lipoproteins (HDL), 1 participates in the early steps of cholesterol efflux from cells and plays a central role in the subsequent steps of the reverse cholesterol transport (RCT) process that removes excess cholesterol from peripheral tissues for excretion or recycling (2). Apolipo- protein A-I (apoA-I) is the main protein component of HDL. The polypeptide chain is arranged in a globular N-terminal domain (residues 1-43) and a C-terminal lipid-binding domain (residues 44-243). There are eight 22-mer and two 11-mer tandem amino acid sequence repeats, each with the periodicity of an amphipathic R-helix (3). ApoA-I binds to the periphery of lipid bilayer disks and to the surface of spherical lipoproteins and synthetic lipid microemulsions (1). On the basis of thermodynamic and circular dichroism measurements, lipid-free apoA-I has been proposed to exhibit a molten globule-like state under physiological conditions (4). Upon lipid binding, the protein R-helix content increases from ∼50% to ∼78% depending on the nature of the lipid bilayer. The roles that different amphipathic R-helices play in the interaction of apoA-I with lipids have been reviewed by Frank and Marcel (5). While the N-terminal helix 44- 65 and the C-terminal helix 210-241 are important for the initial association with lipids, helices 100-121 and 122- 143 are important for lipid binding and HDL maturation. The two helices between residues 144 and 186 contribute less to lipid binding. The central region (residues 87-112) was also found to interact specifically with phosphatidyl- choline (PC) liposomes (6). The structural arrangement of apoA-I in discoidal HDL has been modeled, depicting the protein as having eight amphipathic R-helices connected by -turns (7, 8). The aim of this work is to understand how the interaction of apoA-I with cell membranes is influenced by the presence of cholesterol. In a previous work we examined, using gel electrophoresis and two-photon fluorescence microscopy, the effect of three distinct conformations of apoA-I on its ability to bind and extract lipids from POPC membrane vesicles (9). We found that apoA-I binds reversibly to the vesicles with high affinity but does not extract significant amounts of lipid nor perturb the vesicle structure under the experi- mental conditions used. In the present work, we studied the interaction of lipid-free apoA-I with vesicles of POPC containing varying amounts of cholesterol using two different approaches, isothermal titration calorimetry (ITC) and cir- † This research was supported by National Institutes of Health Grant HL-16059 to A.J. and Fundacio ´n Antorchas Grant 14022/124 to M.A.T. and C.A. * To whom correspondence should be addressed at the Unidad de Biofı ´sica, Universidad del Paı ´s Vasco, P.O. Box 644, E-48080 Bilbao, Espan ˜a. E-mail: gbbarxxc@lg.ehu.es; arnulphi@hotmail.com. ‡ University of Illinois at Urbana-Champaign. § DePaul University. 1 Abbreviations: apoA-I, apolipoprotein A-I; POPC, 1-palmitoyl- 2-oleoylphosphatidylcholine; FC, free cholesterol; SUVs, small unila- mellar vesicles; HDL, high-density lipoproteins; PC, phosphatidylcho- line; ITC, isothermal titration calorimetry; CD, circular dichroism spectroscopy; RCT, reverse cholesterol transport. 12258 Biochemistry 2004, 43, 12258-12264 10.1021/bi036118k CCC: $27.50 © 2004 American Chemical Society Published on Web 09/01/2004