Phospholipid binding and lecithin-cholesterol acyltransferase activation properties of apolipoprotein A-I mutants

Biochemistry. 1995 Oct 17;34(41):13334-42. doi: 10.1021/bi00041a009.

Abstract

Recombinant human apolipoprotein A-I (apo A-I) and three deletion mutants: apo A-I(delta Leu44-Leu126), apo A-I(delta Glu139-Leu170), and apo A-I(delta Ala190-Gln243), purified from the periplasmic space of Escherichia coli, were studied. The rate of turbidity decrease following mixing of apo A-I(delta Ala190-Gln243) with dimyristoylphosphatidylcholine (DMPC) vesicles at 23 degrees C was 10-fold lower than that of the other apo A-I proteins, confirming that the carboxy-terminal region of apo A-I plays a role in rapid lipid binding. The Stokes radii of reconstituted high-density lipoproteins (rHDL), containing dipalmitoylphosphatidylcholine and cholesterol, were larger for the three apo A-I mutants [6.3 nm for apo A-I(delta Leu44-Leu126), 6.1 nm for apo A-I(delta Glu139-Leu170), and 6.5 nm for apo A-I(delta Ala190-Gln243)] than for intact apo A-I (5.0 nm). The mutant rHDL all contained 4 apo A-I molecules per particle as compared to 2 for intact apo A-I. Circular dichroism measurements revealed 8 alpha-helices per apo A-I molecule, 5 per apo A-I(delta Leu44-Leu126), 6 per apo A-I(delta Glu139-Leu170), and 4 per apo A-I(delta Ala190-Gln243) molecule as compared to predicted values of 8, 5, 6, and 6 alpha-helices, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Apolipoprotein A-I / chemistry*
  • Apolipoprotein A-I / metabolism*
  • Apolipoprotein A-I / pharmacology
  • Base Sequence
  • Cloning, Molecular
  • Enzyme Activation
  • Humans
  • Lipoproteins, HDL / chemistry*
  • Molecular Sequence Data
  • Mutagenesis
  • Mutagenesis, Site-Directed
  • Oligodeoxyribonucleotides
  • Peptide Fragments / chemistry
  • Phosphatidylcholine-Sterol O-Acyltransferase / metabolism*
  • Phospholipids / metabolism*
  • Plasmids
  • Protein Structure, Secondary*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology
  • Restriction Mapping
  • Sequence Deletion
  • Spectrometry, Fluorescence

Substances

  • Apolipoprotein A-I
  • Lipoproteins, HDL
  • Oligodeoxyribonucleotides
  • Peptide Fragments
  • Phospholipids
  • Recombinant Proteins
  • Phosphatidylcholine-Sterol O-Acyltransferase