A Three-Dimensional Molecular Model of Lipid-Free Apolipoprotein A-I Determined by Cross-Linking/Mass Spectrometry and Sequence Threading

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• 作者：R. A. Gangani D. Silva ; George M. Hilliard ; Jianwen Fang ; Stephen Macha ; and W. Sean Davidson
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：March 1, 2005
• 年：2005
• 卷：44
• 期：8
• 页码：2759 - 2769
• 全文大小：412K
• 年卷期：v.44,no.8(March 1, 2005)
• ISSN：1520-4995

文摘

Apolipoprotein (apo) A-I, a 243-residue, 28.1-kDa protein is a major mediator of the reversecholesterol transport (RCT) pathway, a process that may reduce the risk of cardiovascular disease inhumans. In plasma, a small fraction of lipid-free or lipid-poor apoA-I is likely a key player in the firststep of RCT. Therefore, a basic understanding of the structural details of lipid-free apoA-I will be usefulfor elucidating the molecular details of the pathway. To address this issue, we applied the combinedapproach of cross-linking chemistry and high-resolution mass spectrometry (MS) to obtain distanceconstraints within the protein structure. The 21 lysine residues within apoA-I were treated with homobifunctional chemical cross-linkers capable of covalently bridging two lysine residues residing within adefined spacer arm length. After trypsin digestion of the sample, individual peptide masses were identifiedby MS just after liquid chromatographic separation. With respect to the linear amino acid sequence, weidentified 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 wasbuilt for the lipid-free apoA-I monomer based on homology with proteins of similar sequence and knownthree-dimensional structures. The result is the first detailed model of lipid-free apoA-I. It depicts a helicalbundle structure in which the N- and C-termini are in close proximity. Furthermore, our data suggest thatthe self-association of lipid-free apoA-I occurs via C- and N-termini of the protein based on the locationsof six cross-links that are unique to the cross-linked dimeric form of apoA-I.