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- W1999906656 abstract "Nanoscale apolipoprotein bound bilayers (NABBs) are soluble phospholipid bilayers encapsulated by the amphipathic helical protein apolipoprotein A-I (Apo A-I). NABBs, unlike detergent micelles and liposomes, provide a homogenous, native-like membrane environment to study structure-function relationships of membrane proteins. NABBs have previously been employed to study G protein-coupled receptors (GPCRs) such as rhodopsin and CCR5, but the approach was limited by poor expression of Apo A-I and low yields of GPCR incorporation. To address these shortcomings, we have engineered several synthetic genes of Apo A-I derived from zebrafish that express at higher levels in E. coli as compared to older Apo A-I variants. The size of the NABBs can be controlled by changing the length of Apo A-I, an additional advantage that will enable study of receptor oligomerization. We intend to use this platform in conjunction with single molecule fluorescence techniques to study GPCR-ligand interactions, focusing on the chemokine receptors CCR5 and CXCR4. The endogenous ligands of these receptors - including RANTES (CCR5) and CXCL12 (CXCR4) - have been shown to prevent HIV particle entry, but the kinetics and structural determinants of binding are poorly characterized. We show receptor labeling with Alexa 488 and 647 using unnatural amino acid mutagenesis and SNAP-tag technologies followed by incorporation into NABBs. The assemblies have been characterized by fluorescence correlation spectroscopy. Future experiments will use multicolor total internal reflection fluorescence (TIRF) microscopy and fluorescence cross-correlation spectroscopy (FCCS) to determine the kinetics of ligand binding and the stoichiometry of the signaling complex. These parameters will shed light on the mechanism of class A GPCR activation and could potentially inform future development of HIV entry blockers." @default.
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- W1999906656 date "2013-01-01" @default.
- W1999906656 modified "2023-09-29" @default.
- W1999906656 title "Incorporation of Fluorescently Tagged Chemokine Receptor 5 (CCR5) into Membrane Nanoparticles" @default.
- W1999906656 doi "https://doi.org/10.1016/j.bpj.2012.11.668" @default.
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