title: Structural analysis of apolipoprotein A-I: limited proteolysis of methionine-reduced and -oxidized lipid-free and lipid-bound human apo A-I.
abstract: The domain structures of lipid-free and lipid-bound apolipoprotein A-I (apo A-I) containing reduced and oxidized methionines were analyzed by limited proteolysis. Lipid-free apo A-I is cleaved primarily in the extreme carboxy-terminus and, to a much lesser extent, in the central region of the protein between residues 115 and 136. Oxidation of methionines 112 and 148 to the corresponding sulfoxides in putative amphipathic helices 4 (P99-E120) and 6 (P143-A164), respectively, causes helices 1 (L44-G65), 2 (P66-S87), and 7 (P165-G186) to become susceptible to protease digestion. These results are consistent with a discrete, globular tertiary structure for the lipid-free protein minimally formed from amphipathic helices 1, 2, 4, 6, and 7. In distinct contrast to lipid-free apo A-I, lipid-bound apo A-I is most susceptible to cleavage in the extreme amino-terminus and, to a lesser extent, in both the central and carboxy-terminal regions. The observed cleavage pattern for the reduced lipid-bound protein supports the existence of many of the turns between helices predicted by sequence analysis of the lipid-bound protein. Methionine oxidation of lipid-bound protein results in a decreased protease susceptibility in the extreme amino-terminus and a concomitant increase in protease susceptibility in the central and carboxy-terminal regions. The results from methionine oxidation indicate the oxidation state of the protein is an important determinant in defining the conformation of both lipid-free and lipid-bound apo A-I.
authors.affiliation: Department of Biological Chemistry, Southern Research Institute, Birmingham, Alabama 35255-5305, USA.
authors.forename: L M
authors.initials: LM
authors.lastname: Roberts
authors.forename: M J
authors.initials: MJ
authors.lastname: Ray
authors.forename: T W
authors.initials: TW
authors.lastname: Shih
authors.forename: E
authors.initials: E
authors.lastname: Hayden
authors.forename: M M
authors.initials: MM
authors.lastname: Reader
authors.forename: C G
authors.initials: CG
authors.lastname: Brouillette
mesh_terms: D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D050356:Lipid Metabolism; D008715:Methionine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000378:metabolism
chemical_list: D016632:Apolipoprotein A-I; D008715:Methionine; D010450:Endopeptidases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase