Faculty Collaboration Database

Intermolecular contact between globular N-terminal fold and C-terminal domain of ApoA-I stabilizes its lipid-bound conformation: studies employing chemical cross-linking and mass spectrometry. J Biol Chem 2005 Sep 23;280(38):33015-25

Date

06/24/2005

Pubmed ID

15972827

DOI

10.1074/jbc.M505081200

Scopus ID

2-s2.0-25444491569 (requires institutional sign-in at Scopus site)   88 Citations

Abstract

The structure of apoA-I on discoidal high density lipoprotein (HDL) was studied using a combination of chemical cross-linking and mass spectrometry. Recombinant HDL particles containing 145 molecules of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and two molecules of apoA-I with a 96-A diameter were treated with the lysine-specific cross-linker, dithiobis(succinimidylpropionate) at varying molar ratios from 2:1 to 200:1. At low molar ratios of dithiobis(succinimidylpropionate) to apoA-I, two products were obtained corresponding to approximately 53 and approximately 80 kDa. At high molar ratios, these two products merged, yielding a product of approximately 59 kDa, close to the theoretical molecular mass of dimeric apoA-I. To identify the intermolecular cross-links giving rise to the two different sized products, bands were excised from the gel, digested with trypsin, and then analyzed by liquid chromatography-electrospray-tandem mass spectrometry. In addition, tandem mass spectrometry of unique cross-links found in the 53- and 80-kDa products suggested that a distinct conformation exists for lipid-bound apoA-I on 96-A recombinant HDL, emphasizing the inherent flexibility and malleability of the N termini and its interaction with its C-terminal domain.

Author List

Bhat S, Sorci-Thomas MG, Alexander ET, Samuel MP, Thomas MJ

Authors

Mary Sorci Thomas PhD Professor in the Medicine department at Medical College of Wisconsin
Michael 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 Sequence
Apolipoprotein A-I
Chromatography, Liquid
Cross-Linking Reagents
Cysteine
Dimerization
Electrophoresis, Polyacrylamide Gel
Glycerylphosphorylcholine
Lipids
Lysine
Mass Spectrometry
Models, Molecular
Molecular Sequence Data
Mutation
Oligonucleotides
Peptides
Protein Conformation
Protein Denaturation
Protein Folding
Protein Structure, Tertiary
Spectrometry, Mass, Electrospray Ionization
Succinimides
Trypsin
Urea