Studies of integral membrane proteins: Membrane assembly of artificial membrane proteins and characterization of an intracellular domain of the human cannabinoid receptor 1
Date of Completion
Biology, Molecular|Chemistry, Biochemistry
Integral membrane proteins (IMPs) comprise about thirty percent of all known proteins, yet comparatively little is known about them relative to soluble proteins. How IMPs assume their three dimensional structures, what those structures are, and how they function in the disparate environs of lipid bilayers and the aqueous juxtamembrane milieu are subjects of much interest. ^ Many proteins, including IMPs, must cross a membrane on their way to secretion or integration. Such trafficking is in many cases performed by a set of proteins that have been termed the translocation machinery. Part of the paucity of information about IMPs is whether and how certain types utilize the translocation machinery. ^ G protein-coupled receptors (GPCRs) are IMPs that have wide ranging roles in cell signaling for coordination of growth, perception, metabolism, and more. The human cannabinoid receptor 1 (CB1) is a GPCR through which Δ 9-tetrahydrocannabinol and other cannabinoids exert pharmacological effects including analgesia, anti-inflammation, and immunosupression. The structural features of CB1 and its interactions with G proteins are of importance in understanding cannabinoid biochemistry, developing therapeutics, and adding to our overall knowledge of GPCR and IMP structure. ^ This is a thesis in two parts. Part I describes experiments in which I determined that, contrary to earlier work, a type III membrane protein utilizes the secretion machinery of Escherichia coli to integrate into the membrane. I converted alkaline phosphatase, a water soluble protein, into different types of IMPs by insertion of artificial transmembrane segments (TMSs) and varying sequences flanking the TMS. The roles of transport machinery components in assembly of the resultant IMPs were determined. Part II consists of work done to develop structural information about CB1, both in its resting and G protein-bound forms. I developed methods for expression and purification of Gαi1 protein and peptides representing intracellular loop 3 (IC3) of the receptor. Wild-type IC3 peptide was labeled with 15 N. Collaborators determined its structure by nuclear magnetic resonance (NMR) spectroscopy and modeled its interaction with membranes. Additionally, Gαi1 was used in transfer nuclear Overhauser effect spectroscopy (trNOESY) with small IC3 peptides to determine their Gαi1-bound structures. ^
McMurry, Jonathan Lawrence, "Studies of integral membrane proteins: Membrane assembly of artificial membrane proteins and characterization of an intracellular domain of the human cannabinoid receptor 1" (2002). Doctoral Dissertations. AAI3062089.