Date of Award
apo A-I interactions, g protein coupled receptors (GPCRs), nanoscale apolipoprotein bound bilayers (NABBs), GPCR stoichiometry, rhodospin
From first principles of phospholipid â€“ apolipoprotein A-I (apo A-I) interactions, we hypothesized that the amino acid sequence of apo A-I derived from a different species may exhibit improved properties compared to human apo A-I for the purpose of incorporation of G protein-coupled receptors (GPCRs) into homogeneous discoidal lipoprotein particles. We generated apolipoprotein A-I DNA derived from zebrafish (Danio rerio) using molecular cloning techniques and optimized a heterologous bacterial expression system, and protein purification and labeling scheme for obtaining high-yields of pure zebrafish apo A-I. We demonstrated that zebrafish apo A-I forms stable, homogeneous discoidal lipoprotein particles, which we termed as Nanoscale Apolipoprotein Bound Bilayers (NABBs). Using bovine rhodopsin as a model system, we developed a novel method of rapidly incorporating GPCRs into NABBs â€“ a significant improvement over traditional methods â€“ requiring less time and materials for receptor reconstitution. The method was generalized for incorporation of heterologously expressed GPCRs in mammalian cells available at comparatively lower levels of receptor concentration and purity. A novel ELISA technique was developed for high-throughput quantification of the incorporated GPCR in NABBs. We also developed methods to control the ratio of receptor to NABB and imaged the rhodopsin-NABBs using electron microscopy (EM) to measure stoichiometry and receptor orientation. Using a combination of EM imaging and biochemical analyses, we correlated stability and signaling efficiency of rhodopsin in NABBs with either one or two receptors. We discovered that the specific activity of G protein coupling for single rhodopsins sequestered in individual NABBs was nearly identical to that of two rhodopsins per NABB under conditions where stoichiometry and orientation could be inferred by electron microscopy imaging. Thermal stability of rhodopsin and CCR5 in NABBs was superior to the receptors in various detergents commonly used for membrane protein work. CCR5 in NABBs retained its ability to activate G protein. This work highlights the NABBs as a promising tool for in-vitro manipulation of GPCR stoichiometry and biophysical studies of GPCRs in an isolated, native-like, cell-free system.
Banerjee, Sourabh, "Studies of G-Protein Coupled Receptors Incorporated Into a Novel, Nanoscale, Membrane-Mimetic System" (2009). Student Theses and Dissertations. 104.