Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy.
Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.
Zhang
Lei
L
Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Song
James
J
Cavigiolio
Giorgio
G
Ishida
Brian Y
BY
Zhang
Shengli
S
Kane
John P
JP
Weisgraber
Karl H
KH
Oda
Michael N
MN
Rye
Kerry-Anne
KA
Pownall
Henry J
HJ
Ren
Gang
G
D016632:Apolipoprotein A-I / Q000097:blood / Q000648:ultrastructure; D053327:Apolipoprotein E4 / Q000097:blood / Q000648:ultrastructure; D006801:Humans; D008074:Lipoproteins / Q000097:blood / Q000648:ultrastructure*; D008075:Lipoproteins, HDL / Q000097:blood / Q000648:ultrastructure; D008854:Microscopy, Electron / Q000379:methods*; D016624:Negative Staining
D016632:Apolipoprotein A-I; D053327:Apolipoprotein E4; D008074:Lipoproteins; D008075:Lipoproteins, HDL