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.
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.
Pathology and Biochemistry, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157, USA.
Shaila
S
Bhat
Mary G
MG
Sorci-Thomas
Eric T
ET
Alexander
Michael P
MP
Samuel
Michael J
MJ
Thomas
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry*; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005997:Glycerylphosphorylcholine / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008239:Lysine / Q000494:pharmacology; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D013388:Succinimides / Q000494:pharmacology; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D016632:Apolipoprotein A-I; D003432:Cross-Linking Reagents; D008055:Lipids; D009841:Oligonucleotides; D010455:Peptides; D013388:Succinimides; D005997:Glycerylphosphorylcholine; D014508:Urea; D014357:Trypsin; C011240:dithiobis(succinimidylpropionate); D008239:Lysine; D003545:Cysteine