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. 2007 Jun 1;92(11):4097-108.
doi: 10.1529/biophysj.106.101105. Epub 2007 Mar 16.

Structural analysis of reconstituted lipoproteins containing the N-terminal domain of apolipoprotein B

Zhenghui Gordon Jiang  1 , Martha N Simon, Joseph S Wall, C James McKnight
Affiliations

Structural analysis of reconstituted lipoproteins containing the N-terminal domain of apolipoprotein B

Zhenghui Gordon Jiang et al. Biophys J. .

Abstract

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.

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Figures

FIGURE 1
FIGURE 1
Homology model of B17. (a) Comparison of domain structures of lipovitellin (LV) and apoB. Only the N-terminal 20% of apoB is colored by proposed domains. β-barrel domain, green; α-helical domain, cyan; C-sheet, red; A-sheet, dark blue; missing regions in the structure of LV, white. A domain diagram of B17 has been colored by phospholipid remodeling efficiencies. Lighter color corresponds to more efficient phospholipid remodeling activities as determined previously (24). (b) Ribbon representation of the B17 model colored by domain as in panel a. (c) Ribbon representation of B17 colored by the phospholipid remodeling sequences as in panel a.
FIGURE 2
FIGURE 2
B6.4-17 forms reconstituted particles with DMPC. (a) Size exclusion chromatography of reconstituted B6.4-17/DMPC particles. Thin shaded line, lipid-free B6.4-17; thick shaded line, B6.4-17/DMPC complex at 1:1 L/P weight ratio; thick solid line, B6.4-17/DMPC complex at 4:1 L/P weight ratio; dotted shaded line, B6.4-17/DMPC complex at 0.8:1 L/P weight ratio with Ni-NTA-Nanogold labels (AU); B6.4-17/DMPC complex at 4:1 L/P weight ratio with Ni-NTA-Nanogold labels. (b,c) Negative stain electron microscopic images of reconstituted B6.4-17/DMPC particles at 1:1 L/P weight ratio (b) and 4:1 L/P weight ratio (c). The magnification bar corresponds to 50 nm in both images.
FIGURE 3
FIGURE 3
Circular dichroism studies of B6.4-17 and its subdomains. (a) CD wavelength scans of B6.4-13 (⋄), B6.4-10 (◃), and B9-13 (▹). Samples contained ∼5 μM protein in 5 mM potassium phosphate at pH 7.5, in a 1 mm cuvette at 25°C. Four scans with an averaging time of 5 s at every nm were collected and the average data are reported. (b) GuHCl titration of B6.4-13 (⋄), B6.4-10 (◃), and B9-13 (▹). (c) GuHCl titration of B6.4-17 (▵) and B6.4-17/DMPC complex (▴) at 1.2:1 L/P ratio. Native proteins in a 1 cm cuvette were titrated by the protein at the same concentration in 7 M GuHCl at 0.1 M per step at 25°C. After each injection of the denaturant, the sample was stirred for 3 min and the CD signal at 222 nm was averaged for 20 s. The raw CD data were converted to percentage unfolded by calculating the ratio between each data point and the CD signal at 0 M GuHCl as described in Materials and Methods. In the inset figure, the percentage unfolded curve was calculated by applying a folded baseline using the first 10 data points and using the CD signal at 6 M GuHCl as the unfolded reference.
FIGURE 4
FIGURE 4
Limited proteolysis of B6.4-17 and B6.4-17/DMPC particles. Lipid-free B6.4-17 and B6.4-17/DMPC complexes (1.2:1 wt/wt L/P ratio) were prepared at 1 mg/ml protein concentration in 20 mM Tris-HCl and 150 mM sodium chloride, pH 7.5. Freshly prepared Trypsin in 1 mM HCl was added to a protein to trypsin ratio of 1000:1 (wt/wt) and incubated for 15 min at room temperature. The digestion was stopped by the addition of 10 mM β-mercaptoethanol and acetic acid to 5%. Lane 1, protein standards; lane 2, B6.4-17 before digestion; lane 3, B6.4-17 after digestion; lane 4, B6.4-17/DMPC after digestion. The major proteolytic products are labeled to the left of the gel and are identified in Table 1.
FIGURE 5
FIGURE 5
Imaging of the reconstituted B6.4-17/DMPC particles. (a) Negative stain image of reconstituted DMPC particles labeled with Ni-NTA-Nanogold. (b) Negative stain image of reconstituted DMPC particles labeled with Ni-NTA-Nanogold after gold enhancement. Ni-NTA-Nanogold was added to a 3:1 molar ratio to the protein/lipid mixture before the preparation of EM-grids. Protein samples were stained by Nanovan and gold enhancement was performed according to the protocol from Nanoprobes. The magnification bar corresponds to 20 nm for both images. White triangles point to gold particles. (c) STEM image of reconstituted B6.4-17/DMPC particles at 0.8:1 L/P weight ratios. The reconstituted particles had been purified on a Superdex GL 200 column (Fig. 2). Tobacco mosaic virus was used as an internal standard as shown in the image. The magnification bar corresponds to 20 nm. Six reconstituted particles (i–vi) are shown in enlarged views (36 × 36 nm) on the right.
FIGURE 6
FIGURE 6
Categories of nanogold-labeled particles. (a) Histogram of the number of nanogold on each particle. A total of 1315 particles were analyzed, and are categorized based on the number of nanogolds attached on each particle. (b) Accumulated plot of the nanogold count. Each data point represents the sum of the number of particles in this group plus the accumulated number of particles in the previous groups. (c) Histogram of the 16 geometrical groups. Nanogold-labeled particles were categorized into 16 geometrical groups as indicated at the bottom of the histogram. The number of particles counted in each group was plotted with shaded bars.
FIGURE 7
FIGURE 7
Molecular weights of nanogold-labeled particles. (a) The average molecular weight of the reconstituted particle when different numbers of nanogold labels are present. The molecular weight of each reconstituted particle was measured with PCMass28 (30). The mass that corresponds to the nanogold was excluded during statistical analysis. The error bar corresponds to the standard deviation of the calculated molecular weight at each stoichiometry. The number of particles in each group was labeled on the top of each dataset. Datasets with an asterisk have a P-value <0.01 among each other, as calculated by analysis of variance in Origin 7.5 (Microcal). (b,c) The frequency of occurrence of the molecular weight for particles with a C2 symmetry (b) or a C3 symmetry (c).
FIGURE 8
FIGURE 8
Models of reconstituted B6.4-17/DMPC particles. (a) Domain diagram of B6.4-17. The N-terminal half of the α-helical domain, green; C-terminal half of the α-helical domain, cyan; β-sheets in the C-sheet domain, red; proposed α-helical region in the C-sheet domain missing in the lipovitellin crystal structure, magenta. (b) Proposed domain interactions in lipid-bound B6.4-17. The black sphere indicates a nanogold label. (c) A three-dimensional cartoon of B6.4-17 in a discoidal particle. The orange disks represent a DMPC bilayer. The lipid core and B6.4-17 domains are shown approximately to scale. (d–f) Proposed protein assemblies in the B6.4-17/DMPC particle. (d) Two proteins in a head-to-tail assembly. (e) Two proteins in a head-to-head assembly. (f) Three proteins in a symmetric assembly. The expected STEM views are shown on the right corner of each model.
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