The interaction of human apolipoprotein C-I with sub-micellar phospholipid.
Mature human apolipoprotein C-I (apoC-I), comprising 57 amino acids, is the smallest member of the plasma apolipoprotein family. Amphipathic helical regions within apoC-I, common to this class of proteins, are mediators of lipid binding, a process that underlies the functional properties of apoC-I, including the capacity to activate the plasma enzyme LCAT, to disrupt apoE mediated receptor interactions and to inhibit cholesterol ester transfer protein. To examine apoC-I/phospholipid interactions, we have developed an expression system in Escherichia coli to obtain purified apoC-I with yields of approximately 4-5 mg per L of culture. The purified product has properties similar to plasma-derived apoC-I including self-association in the lipid-free state and induced alpha-helical content in the presence of egg-yolk phosphatidylcholine and dimyristoylglycerophosphocholine vesicles. We chose the short-chain phospholipid, dihexanoylglycerophosphocholine (Hex2Gro-PCho), to examine the interaction of apoC-I with submicellar phospholipid. Circular dichroism spectroscopy and cross-linking experiments show that apoC-I acquires helical content and remains self-associated at submicellar concentrations of Hex2Gro-PCho (4 mM). Sedimentation equilibrium studies of apoC-I at submicellar levels of Hex2Gro-PCho and analysis of the effects of apoC-I on the 1H NMR spectrum of Hex2Gro-PCho indicate micelle induction by apoC-I, and establish the capacity of apoC-I to assemble individual phospholipid molecules.
Atcliffe
B W
BW
Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Victoria, Australia.
MacRaild
C A
CA
Gooley
P R
PR
Howlett
G J
GJ
D053302:Apolipoprotein C-I; D001056:Apolipoproteins C / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D050356:Lipid Metabolism; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D010713:Phosphatidylcholines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D014461:Ultracentrifugation
C092445:1,2-dihexanoyl-sn-glycero-3-phosphorylcholine; D053302:Apolipoprotein C-I; D001056:Apolipoproteins C; D003432:Cross-Linking Reagents; D008823:Micelles; D010713:Phosphatidylcholines; D010743:Phospholipids; D004134:Dimyristoylphosphatidylcholine