SUPRAMOLECULAR ASSEMBLIES |
Structural Analysis of Reconstituted Lipoproteins
Containing the N-terminal Domain of Apolipoprotein B
Zhenghui Gordon Jiang 1, Martha N, Simon 2, Joseph S. Wall 2 and C. James McKnight 1*
1 Boston University School of Medicine
2 Brookhaven National Laboratory
* To whom correspondence should be addressed. E-mail: cjmck{at}bu.edu.
Submitted on November 16, 2006
Revised on December 28, 2006
Accepted on 19 January 2007
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Abstract |
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Apolipoproteins play a central role in lipoprotein metabolism, and are directly implicated in cardiovascular diseases, but their structural characterization has been complicated by their structural flexibility and heterogeneity. Here we describe the structural characterization of the N terminal region of apolipoprotein B (apoB), the major protein component of very low density lipoprotein and low density lipoprotein, in the presence of phospholipids. Specifically, we focus on the N-terminal 6.4-17% of ApoB (B6.4-17) complexed with the phospholipid dimyristoylphosphatidylcholine (DMPC), in vitro. In addition to circular dichroism spectroscopy (CD) and limited proteolysis, our strategy incorporates nanogold-labeling of the protein in the reconstituted lipoprotein complex followed by visualization and molecular weight determination with scanning transmission electron microscopy imaging (STEM). Based on the STEM analysis of ~1300 individual particles where the B6.4-17 is labeled with nanogold through a six-his tag, most complexes contain either two or three B6.4-17 molecules. CD and limited proteolysis of these reconstituted particles indicate that there are no large conformational changes in B6.4-17 upon lipoprotein complex formation. This is in contrast to the large structural changes that occur during apolipoprotein A-I-lipid interactions. The method described here allows a direct measurement of the stoichiometry and molecular weight of individual particles, rather than the average of the entire sample. Thus, it represents a useful strategy to characterize the structure of lipoproteins, which are not structurally uniform, but can still be defined by an ensemble of related patterns.
Key Words:
Apolipoprotein B, dimyristoylphosphatidylcholine, low density lipoprotein, nanogold, reconstituted lipoprotein particle, scanning transmission electron microscopy
