Structural characterization of the membrane-targeting domain of regulator of G protein signaling 4

Signal transduction pathways mediated by G proteins are important regulators of neuronal function. Heterotrimeric G proteins transduce and amplify signals from activated receptors to effector molecules. The duration and strength of an activated G-protein signal is modulated by a variety of mechanisms. One form of regulation comes from a family of RGS (R&barbelow;egulators of G&barbelow; protein S&barbelow;ignaling) proteins. These proteins inhibit signaling through G-protein regulated pathways by acting as GTPase activating proteins. In order to interact with their G-protein substrates, RGS proteins must localize to the plasma membrane. Previous work in the Linder lab established that RGS4, found in neural and cardiac tissue, has an intrinsic ability to bind to membranes through its amino terminal thirty-three amino acids. This thesis tests the hypothesis that the amino terminal membrane-targeting domain of RGS4 mediates membrane binding through the formation of an amphipathic alpha-helix.; The structural biology techniques of circular dichroism (CD) spectroscopy and multidimensional nuclear magnetic resonance (NMR) were used to examine the structural characteristics of the membrane-targeting domain of RGS4 in solution and in the presence of membrane mimics. A synthetic peptide corresponding to the first 31 amino acid residues of the membrane-targeting domain of RGS4 was examined by CD spectroscopy. Samples were analyzed in aqueous solution alone, or in the presence of lipid vesicles or detergent micelles. In solution alone, the peptide adopts a random coil conformation and converts to an alpha-helical conformation in the presence of membrane mimics. The membrane-targeting domain of RGS4, in the context of the full-length protein, was further studied by NMR. To produce RGS4 enriched with 13C and 15N isotopes, untagged RGS4 was expressed in E. coli and purified using ion exchange and gel filtration chromatography. Data were collected in the absence and presence of small detergent micelles. Backbone assignments were made for the N- and C-termini of RGS4. Comparisons were made between the observed chemical shifts of the termini with standard random coil chemical shifts. The N-terminus of RGS4 undergoes a transition between disordered and ordered states corresponding to random coil and alpha-helical conformations. In solution, the N-terminus favors the random coil state. Upon binding to detergent micelles it favors an alpha-helical-like conformation from residues 10 through 32. The C-terminus of RGS4 remains in a random coil conformation in the presence or absence of detergent micelles. These findings support a model where the N-terminus of RGS4 is able to sample multiple conformational states. The ability of the N-terminus to adopt multiple conformations may be important for receptor-RGS interactions.