Conformation of dimeric apolipoprotein A-I milano on recombinant lipoprotein particles. Biochemistry 2010 Jun 29;49(25):5213-24
Date
06/08/2010Pubmed ID
20524691Pubmed Central ID
PMC2946800DOI
10.1021/bi1003734Scopus ID
2-s2.0-77953891139 (requires institutional sign-in at Scopus site) 16 CitationsAbstract
Apolipoprotein A-I Milano (apoA-I(Milano)) is a naturally occurring human mutation of wild-type apolipoprotein A-I (apoA-I(WT)) having cystine substituted for arginine(173). Two molecules of apo-I(WT) form disks with phospholipid having a defined relationship between the apoA-I(WT) molecules. ApoA-I(Milano) forms cystine homodimers that would not allow the protein to adopt the conformation reported for apoA-I(WT). The conformational constraints for dimeric apoA-I(Milano) recombinant high-density lipoprotein (rHDL) disks made with phospholipid were deduced from a combination of chemical cross-linking and mass spectrometry. Lysine-selective homobifunctional cross-linkers were reacted with homogeneous rHDL having diameters of 78 and 125 A. After reduction, cross-linked apoA-I(Milano) was separated from monomeric apoprotein by gel electrophoresis and then subjected to in-gel trypsin digest. Cross-linked peptides were confirmed by MS/MS sequencing. The cross-links provided distance constraints that were used to refine models of lipid-bound dimeric apoA-I(Milano). These studies suggest that a single dimeric apoA-I(Milano) on 78 A diameter rHDL girdles the edge of a phospholipid disk assuming a "belt" conformation similar to the "belt" region of apoA-I(WT) on rHDL. However, the C-terminal end of dimeric apoA-I(Milano) wraps around the periphery of the particle to shield the fatty acid chains from water rather than folding back onto the "belt" as does apoA-I(WT). The two apoA-I(Milano) dimers on a 125 A diameter rHDL do not encircle the periphery of a phospholipid disk but appear to reside on the surface of a laminar micelle.
Author List
Bhat S, Sorci-Thomas MG, Calabresi L, Samuel MP, Thomas MJAuthors
Mary Sorci Thomas PhD Professor in the Medicine department at Medical College of WisconsinMichael J. Thomas PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Amino Acid SequenceApolipoprotein A-I
Dimerization
Electrophoresis, Polyacrylamide Gel
Humans
Mass Spectrometry
Molecular Sequence Data
Protein Conformation
Recombinant Proteins