Vesicle-reconstituted low density lipoprotein receptor. Visualization by cryoelectron microscopy.
The low density lipoprotein (LDL) receptor is a key protein for maintaining cellular cholesterol homeostasis by binding cholesterol-rich lipoproteins through their apoB and apoE apoproteins. The LDL receptor is a transmembrane glycoprotein of M(r) approximately 115 kDa; based on its primary sequence, five distinct structural domains have been identified (Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey, M. L., Goldstein, J. L., and Russell, D. W. (1984) Cell 39, 27-38). As a first step toward providing a structural description of the intact LDL receptor, the receptor has been purified from bovine adrenal cortices, reconstituted into unilamellar egg yolk phosphatidylcholine vesicles, and imaged using cryoelectron microscopy (cryoEM). CryoEM has the advantage of providing images of the reconstituted LDL receptor in its frozen, fully hydrated state. LDL receptor molecules were visualized as elongated, stick-like projections from the vesicle surface with maximum dimensions approximately 120-A length by approximately 45-A width. In some of the images, a short arm (or arms) was visible at the distal end of the stick-like projections. The LDL receptor was labeled via accessible free cysteine residues, probably including that corresponding to Cys-431 of the known full-length sequence of the human LDL receptor. The accessible cysteine was demonstrated using a maleimide-biotin.streptavidin conjugate and confirmed by labeling with monomaleimido-Nanogold. Images obtained by cryoEM showed that the extracellular stick-like domain of the reconstituted LDL receptor was labeled by Nanogold. This combined cryoEM-Nanogold labeling study has provided the first low resolution structural images of the reconstituted, full-length bovine LDL receptor.
Jeon
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Departments of Biophysics and Biochemistry, Center for Advanced Biomedical Research, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Shipley
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GG
D000302:Adrenal Cortex / Q000737:chemistry; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives; D002417:Cattle; D020285:Cryoelectron Microscopy; D006046:Gold; D007091:Image Processing, Computer-Assisted; D008081:Liposomes; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D010713:Phosphatidylcholines; D011973:Receptors, LDL / Q000648:ultrastructure*; D013194:Staining and Labeling; D019809:Streptavidin / Q000031:analogs & derivatives
D008081:Liposomes; D008301:Maleimides; D010713:Phosphatidylcholines; D011973:Receptors, LDL; C043592:maleimide; D001710:Biotin; D006046:Gold; D019809:Streptavidin