Lipid binding of human apolipoprotein A-I (apoA-I) occurs initially through the C-terminal α-helices followed by conformational reorganization of the N-terminal helix bundle. This led us to hypothesize that apoA-I has multiple lipid-bound conformations, in which the N-terminal helix bundle adopts either open or closed conformations anchored by the C-terminal domain. To investigate such possible conformations of apoA-I at the surface of a spherical lipid particle, site-specific labeling of the Nand C-terminal helices in apoA-I by N-(1-pyrene)maleimide was employed after substitution of a Cys residue for Val-53 or Phe-229. Neither mutagenesis nor the pyrene labeling caused discernible changes in the lipid-free structure and lipid interaction of apoA-I. Taking advantage of a significant increase in fluorescence when a pyrene-labeled helix is in contact with the lipid surface, we monitored the behaviors of the N-and C-terminal helices upon binding of apoA-I to egg PC small unilamellar vesicles. Comparison of the binding isotherms for pyrene-labeled apoA-I as well as a C-terminal helical peptide suggests that an increase in surface concentration of apoA-I causes dissociation of the N-terminal helix from the surface leaving the C-terminal helix attached. Consistent with this, isothermal titration calorimetry measurements showed that the enthalpy of apoA-I binding to the lipid surface under near saturated conditions is much less exothermic than that for binding at a low surface concentration, indicating the N-terminal helix bundle is out of contact with lipid at high apoA-I surface concentrations. Interestingly, the presence of cholesterol significantly induces the open conformation of the helix bundle. These results provide insight into the multiple lipid-bound conformations that the N-terminal helix bundle of apoA-I can adopt on a lipid or lipoprotein particle, depending upon the availability of space on the surface and the surface composition.High density lipoprotein (HDL) is of great clinical importance because elevated levels of plasma HDL cholesterol are associated with a reduced incidence of coronary artery disease (1,2). Apolipoprotein A-I (apoA-I) is the principal protein in HDL and it plays a central role in HDL metabolism (3). The protective functions of HDL and apoA-I against coronary artery disease are due in part to their participation in reverse cholesterol transport, a process by which cholesterol in peripheral cells is transferred via HDL to the liver for catabolism (4,5). The ability of apoA-I to bind to lipid membranes, receptors, and ATP-binding cassette (ABC) transporters at cell surfaces is central to mediating the transport of cholesterol into and out of cells (6)(7)(8) ApoA-I is a 243-residue polypeptide that contains characteristic 11-and 22-residue repeats of amphipathic α-helices (9). The N-and C-terminal helical regions in the apoA-I molecule contribute to the strong lipid binding properties of this protein (10-12) as well as the conformational stability in solution (13,14). It has b...

Carriers of the apolipoprotein A-I Milano (apoA-I M ) variant, R173C, have reduced levels of plasma HDL but no increase in cardiovascular disease. Despite intensive study, it is not clear whether the removal of the arginine or the introduction of the cysteine is responsible for this altered functionality. We investigated this question using two engineered variations of the apoA-I M mutation: R173S apoA-I, similar to apoA-I M but incapable of forming a disulfide bond, and R173K apoA-I, a conservative mutation. Characterization of the lipid-free proteins showed that the order of stability was wild type≈R173K.R173S.R173C. Compared with wildtype apoA-I, apoA-I M had a lower affinity for lipids, while R173S apoA-I displayed intermediate affinity. The in vivo effects of the apoA-I variants were measured by injecting apoA-I-expressing adeno-associated virus into apoA-I-null mice. Mice that expressed the R173S variant again showed an intermediate phenotype. Thus, both the loss of the arginine and its replacement by a cysteine contribute to the altered properties of apoA-I M . The arginine is potentially involved in an intrahelical salt bridge with E169 that is disrupted by the loss of the positively charged arginine and repelled by the cysteine, destabilizing the helix bundle domain in the apoA-I molecule and modifying its lipid binding