title: Lipid-binding studies of human apolipoprotein A-I and its terminally truncated mutants.
abstract: Apolipoprotein A-I (apoA-I, 243 amino acids) is the major protein of high-density lipoproteins (HDL) that plays an important structural and functional role in lipid transport and metabolism. The central region of apoA-I (residues 60-183) is predicted to contain exclusively amphipathic alpha-helices formed from tandem 22-mer sequence repeats. To analyze the lipid-binding properties of this core domain, four terminally truncated mutants of apoA-I, Delta(1-41), Delta(1-59), Delta(1-41,185-243), and Delta(1-59,185-243), were expressed in baculovirus infected Sf-9 cells. The effects of mutations on the ability of apoA-I to form bilayer disk complexes with dimyristoyl phosphatidylcholine (DMPC) that resemble nascent HDL were analyzed by density gradient ultracentrifugation and electron microscopy (EM). The N-terminal deletion mutants, Delta(1-41) and Delta(1-59), showed altered lipid-binding ability as compared to plasma and wild-type apoA-I, and in the double deletion mutants, Delta(1-41, 185-243) and Delta(1-59, 185-243), the lipid binding was abolished. Thermal unfolding of variant apoA-I/DMPC complexes monitored by circular dichroism (CD) showed hysteresis and a shift in the melting curves by about -12 degrees C upon reduction in the heating rate from 1.0 to 0.067 K/min. This indicates an irreversible kinetically controlled transition with a high activation energy E(a) = 60 +/- 5 kcal/mol. CD and EM studies of the apoA-I/DMPC complexes at different pH demonstrated that changes in the net charge or in the charge distribution on the apoA-I molecule have critical effects on the conformation and lipid-binding ability of the protein.
authors: Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118, USA.
authors: Yiling
authors: Y
authors: Fang
authors: Olga
authors: O
authors: Gursky
authors: David
authors: D
authors: Atkinson
mesh_terms: D016632:Apolipoprotein A-I / Q000097:blood / Q000737:chemistry* / Q000235:genetics* / Q000648:ultrastructure; D002867:Chromones / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D014644:Genetic Variation; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D020033:Protein Isoforms / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D017384:Sequence Deletion / Q000235:genetics*; D013816:Thermodynamics
chemical_list: D016632:Apolipoprotein A-I; D002867:Chromones; D046911:Macromolecular Substances; D010446:Peptide Fragments; D020033:Protein Isoforms; D011994:Recombinant Proteins; C066414:5,8-dihydroxy-2-(2-(4-methoxyphenyl)ethyl)chromone