Structural analysis of reconstituted lipoproteins containing the N-terminal domain of apolipoprotein B.
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 in vitro. In addition to circular dichroism spectroscopy 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. Based on the scanning transmission electron microscopy imaging analysis of approximately 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. Circular dichroism spectroscopy 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.
Jiang
Zhenghui Gordon
ZG
Boston University School of Medicine, Department of Physiology & Biophysics, Boston, Massachusetts, USA.
Simon
Martha N
MN
Wall
Joseph S
JS
McKnight
C James
CJ
D001055:Apolipoproteins B / Q000737:chemistry*; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine; D008956:Models, Chemical; D017434:Protein Structure, Tertiary
D001055:Apolipoproteins B; D004134:Dimyristoylphosphatidylcholine