Scavenger receptor class B, type I (SR-BI) homo-dimerizes via its C-terminal region: Fluorescence resonance energy transfer analysis
Autor: | Yolanda F. Darlington, Daisy Sahoo, Yinan Peng, Margery A. Connelly, Jeffery R. Smith |
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Rok vydání: | 2007 |
Předmět: |
Photobleaching
COS cells Chemistry Recombinant Fusion Proteins Cell Biology Scavenger Receptors Class B Fusion protein Article Transport protein Luminescent Proteins Protein Transport Förster resonance energy transfer Biochemistry Cell surface receptor COS Cells Chlorocebus aethiops Fluorescence Resonance Energy Transfer Biophysics Animals Humans Protein quaternary structure Scavenger receptor Dimerization Molecular Biology |
Zdroj: | Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 1771:818-829 |
ISSN: | 1388-1981 |
Popis: | Expression of the scavenger receptor class B, type I (SR-BI) receptor facilitates high density lipoprotein cholesterol transport and correlates with protection against atherosclerosis. Studies have shown that SR-BI self-associates, but many of the techniques used to characterize SR-BI homo-oligomerization were wrought with the prospect of producing artifacts. Therefore, we employed fluorescence resonance energy transfer (FRET) to visualize SR-BI homo-oligomerization with the benefit of gaining information about its quaternary structure in the absence of typical membrane receptor artifacts. To this end, SR-BI was tagged at the N- or C-termini with either cyan (CFP) or yellow (YFP) fluorescent protein. To test whether SR-BI subunits oligomerize through N-N, N-C or C-C terminal interactions, we co-expressed the appropriate SR-BI fusion protein combinations in COS-7 cells and measured live-cell FRET following acceptor photobleaching. We did not observe FRET with co-transfection of SR-BI with CFP and YFP at the N-termini nor at the N- and C-termini, suggesting that the N-termini are not proximal to each other or to the C-termini. However, FRET was observed with co-transfection of SR-BI with CFP and YFP at the C-termini, suggesting that the C-terminal ends are within 10 nm of each other, consistent with SR-BI dimerization via its C-terminal region. |
Databáze: | OpenAIRE |
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