Electrostatic Effects on the Stability of Discoidal High-Density Lipoproteins † Sangeeta Benjwal, Shobini Jayaraman, and Olga Gursky* Department of Physiology and Biophysics, Boston UniVersity School of Medicine, Boston, Massachusetts 02118 ReceiVed April 28, 2005; ReVised Manuscript ReceiVed June 1, 2005 ABSTRACT: High-density lipoproteins (HDL) remove cholesterol from peripheral tissues and thereby help to prevent atherosclerosis. Nascent HDL are discoidal complexes composed of a phospholipid bilayer surrounded by protein R-helices that are thought to form extensive stabilizing interhelical salt bridges. Earlier we showed that HDL stability, which is necessary for HDL functions, is modulated by kinetic barriers. Here we test the role of electrostatic interactions in the kinetic stability by analyzing the effects of salt, pH, and point mutations on model discoidal HDL reconstituted from human apolipoprotein C-1 (apoC-1) and dimyristoyl phosphatidylcholine (DMPC). Circular dichroism, Trp fluorescence, and light scattering data show that molar concentrations of NaCl or Na 2 SO 4 increase the apparent melting temperature of apoC-1:DMPC complexes by up to 20 °C and decelerate protein unfolding. Arrhenius analysis shows that 1 M NaCl stabilizes the disks by δ∆G* = 3.5 kcal/mol at 37 °C and increases the activation energy of their denaturation and fusion by δE a = δ∆H* = 13 kcal/mol, indicating that the salt-induced stabilization is enthalpy-driven. Denaturation studies in various solvent conditions (pH 5.7-8.2, 0-40% sucrose, 0-2 M trimethylamine N-oxide) suggest that the salt-induced disk stabilization results from ionic screening of unfavorable short-range Coulombic interactions. Thus, the dominant electrostatic interactions in apoC- 1:DMPC disks are destabilizing. Comparison of the salt effects on the protein:lipid complexes of various composition reveals an inverse correlation between the lipoprotein stability and the salt-induced stabilization and suggests that short-range electrostatic interactions significantly contribute to lipoprotein stability: the better-optimized these interactions are, the more stable the complex is. Lipoproteins are macromolecular complexes of proteins and lipids that mediate cholesterol transport and metabolism and are central in the pathogenesis of coronary artery disease. High-density lipoproteins (HDL) 1 remove cholesterol from peripheral tissues to the liver for catabolism via the process termed reverse cholesterol transport. Plasma levels of HDL and its major protein, apolipoprotein A-1 (apoA-1), correlate inversely with the probability of developing atherosclerosis (reviewed in refs 1-3). Nascent HDL form discoidal particles composed of a phospholipid bilayer and proteins that are thought to adopt a “double-belt” helical conformation at the disk circumference (4, 5), thereby conferring lipoprotein stability and solubility (Figure 1). HDL proteins belong to a family of exchangeable apolipoproteins. Their amino acid sequences contain 11-mer tandem repeats that have high propensity to form amphipathic R-helices. Class-A R-helices, which are the major lipid-binding motifs in apolipoproteins, have large apolar faces (30-50% of the surface area) that can bind to the phospholipid surface (6). These helices have an unusually high content of charged residues (about 40%) with distinct radial distribution: acidic groups are located in the middle of the polar helical face and basic groups at its edge (Figure 1). This suggests the importance of electrostatic interactions for lipoprotein assembly, structural stability, and functions (4, 7). Electrostatic protein-protein interactions are implicated in several lipoprotein functions. Most notably, charge complementarity is essential for binding of low-density and very low density lipoproteins (LDL and VLDL) to cell receptors (8-10 and references therein). An array of basic groups in helix-4 of apolipoprotein E (apoE) is critically involved in specific binding of VLDL to the acidic site on LDL receptor (8-10); in addition, binding of this array to anionic cell-surface heparin sulfate proteoglycans mediates clearance of lipoprotein remnants (11, 12). Specific electro- static interactions have been proposed to play a role in HDL binding to lipid transfer proteins that mediate lipid exchange among lipoproteins (13, 14), and to lipophilic enzymes such as lecithin:cholesterol acyltransferase (LCAT) whose reaction is central in reverse cholesterol transport (15, 16). Further- more, cholesterol efflux from tissue cells to HDL, which is the first step in reverse cholesterol transport mediated by the ATP-binding cassette transporter ABCA1, is affected by the charge distribution in helix-10 of apoA-1 (17, 18). This suggests that electrostatic interactions affect the apolipopro- tein conformation and/or functional interactions with lipo- protein receptors, lipid transporters, and lipophilic enzymes. † This work is supported by NIH Grant GM67260 to O.G. Electron microscopy, protein spectroscopy, calorimetry, and biochemistry core facilities are supported, in part, by NIH Grant HL26355 (David Atkinson, Program Director). * Author to whom correspondence should be addressed. Mailing address: Department of Physiology and Biophysics, W321, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. E-mail: gursky@bu.edu. Phone: (617) 638-7894. Fax: (617) 638-4041. 1 Abbreviations: HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very low density lipoprotein; apo, apolipoprotein; DMPC, dimyristoyl phosphatidylcholine; DPPC, dipalmitoyl phos- phatidylcholine; LCAT, lecithin:cholesterol acyltransferase; ABCA1, ATP-binding cassette transporter type 1; WT, wild type; CD, circular dichroism; DSC, differential scanning calorimetry; EM, electron microscopy; T-jump, temperature jump. 10218 Biochemistry 2005, 44, 10218-10226 10.1021/bi050781m CCC: $30.25 © 2005 American Chemical Society Published on Web 06/28/2005