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A three-dimensional molecular model of lipid-free apolipoprotein A-I determined by cross-linking/mass spectrometry and sequence threading.

Abstract：

:Apolipoprotein (apo) A-I, a 243-residue, 28.1-kDa protein is a major mediator of the reverse cholesterol transport (RCT) pathway, a process that may reduce the risk of cardiovascular disease in humans. In plasma, a small fraction of lipid-free or lipid-poor apoA-I is likely a key player in the first step of RCT. Therefore, a basic understanding of the structural details of lipid-free apoA-I will be useful for elucidating the molecular details of the pathway. To address this issue, we applied the combined approach of cross-linking chemistry and high-resolution mass spectrometry (MS) to obtain distance constraints within the protein structure. The 21 lysine residues within apoA-I were treated with homo bifunctional chemical cross-linkers capable of covalently bridging two lysine residues residing within a defined spacer arm length. After trypsin digestion of the sample, individual peptide masses were identified by MS just after liquid chromatographic separation. With respect to the linear amino acid sequence, we identified 5 short-range and 12 long-range cross-links within the monomeric form of lipid-free apoA-I. Using the cross-linker spacer arm length as a constraint for identified Lys pairs, a molecular model was built for the lipid-free apoA-I monomer based on homology with proteins of similar sequence and known three-dimensional structures. The result is the first detailed model of lipid-free apoA-I. It depicts a helical bundle structure in which the N- and C-termini are in close proximity. Furthermore, our data suggest that the self-association of lipid-free apoA-I occurs via C- and N-termini of the protein based on the locations of six cross-links that are unique to the cross-linked dimeric form of apoA-I.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Silva RA,Hilliard GM,Fang J,Macha S,Davidson WS

doi

10.1021/bi047717+

subject

Has Abstract

pub_date

2005-03-01 00:00:00

pages

2759-69

issue

8

eissn

0006-2960

issn

1520-4995

journal_volume

44

pub_type

杂志文章

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