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Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pharmacology and Toxicology. Title from title-page of electronic thesis. Bibliography: leaves 130-143.

Thesis (Ph. D.)--University of Virginia, 2001. / Includes bibliographical references (leaves 123-140). Also available online through Digital Dissertations.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: Human A2A adenosine receptors. Includes bibliographical references (115-135). Also available online through Digital Dissertations.

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Neurosciences. Includes bibliographical references. Available online via OhioLINK's ETD Center.

G proteins are alpha-beta-gamma heterotrimers in the resting state, bound to GDP and complexed with the unbound receptor. Once the receptor becomes occupied, the alpha subunit exchanges GDP for GTP, becomes activated, and dissociates from the receptor and can stimulate or inhibit many intracellular activities such as phosphorylation and channel conductance. For example, Gs and Golf alpha subunits stimulate and Gi alpha subunits inhibit adenylyl cyclase. Go alpha subunits are abundant in brain, but are of unknown function. cDNAs for the alpha subunit have been cloned. In order to examine the relative distributions of G proteins in the brain, we used in situ hybridization with radiolabelled synthetic oligonucleotide probes. By using a tyrosine hydroxylase antibody, we found that the dopaminergic neurons of the substantia nigra and the noradrenergic neurons of the locus ceruleus express mRNA for the alpha subunits for each of Gi, Go, and Gs. We noted a paucity of Gs mRNA in the striatum. This was surprising because the basal ganglia contain a dopamine-stimulated adenylyl cyclase activity which has been assumed to be transduced by Gs. Also, immunohistochemistry, immunoblotting, and cholera ADP-ribosylation indicated a very high level of Gs alpha-like protein in the striatum. In order to ascertain which specific G protein we were detecting, we made probes to a new G protein previously identified in the olfactory system. Golf is a stimulatory G protein with size and sequence characteristics similar to those of Gs. The cholera toxin ADP-ribosylation site and C-terminal region to which the antibody was made are identical. We made oligonucloetide probes to the translated and untranslated portions of Golf alpha. High levels Golf mRNA and protein were detected in the striatum and nucleus accumbens, in addition to the expected high levels in the olfactory tubercle. Northern blot studies indicated that Golf transcripts are approximately ten-fold more abundant than Gs alpha transcripts in the striatum. These data indicate that Golf in not an olfactory-specific G protein. It is also the major stimulatory G protein in the basal ganglia. The selective expression of high levels of Golf in dopamine-rich forebrain areas suggest that it may couple DI dopamine receptors to adenylyl cyclase. The role of Golf in dopaminergic neurotransmission and neuropsychiatric disease should be considered. / Medicine, Faculty of / Graduate

Thesis (Ph. D.)--University of Virginia, 2008. / Spine title: Characterization of BAI1. Includes bibliographical references. Also available online through Digital Dissertations as viewed 4/21/2009.

Based on what we have found, we proposed a model for Ni presenting by HypB involved in hydrogenase maturation. HypB binds two Ni ions in the apo- and GDP-bound form. Ni binding also induces dimerization of HypB. Upon GTP binding, HypB can bind an extra Ni ion at the dimeric interface. GTP hydrolysis will release the extra Ni ion, which may be subsequently inserted into hydrogenases during the maturation process. / Furthermore, two Ni binding sites were determined in a monomeric HypB. One is the cluster including C92, H93 and C122, the other is composed of H97 and H101. Upon GTP-dependent dimerization, HypB can bind an extra Ni ion. Our results have shown that the C92/H93/C122 is involved in binding the extra Ni ion, and such binding requires both cysteine residues in the reduced form. Since the GTP-induced dimerization of HypB is coupled to bind an extra Ni, so HypB could act as a GTP-mediated switch that regulate one Ni release from the GTP-bound form to the GDP-bound form. / In the future, we will attempt to crystallize AfHypB in complex with GDP, GTP analogue and AfHypA. Availability of good quality crystals will pave way for the structure determination of AfHypA and AfHypA/HypB complex. And the results obtained will provide a better understanding of the mechanism of functional interaction between HypA and HypB and how HypA and HypB play a role in Ni ion delivery for hydrogenase maturation. / The assembly of the [NiFe]-hydrogenases requires incorporation of Ni ions into the enzyme's metallocenter, which process requires the GTPase activity of HypB and HypA. Due to the essential role in assembly of the active site of hydrogenases, the two proteins were defined as hydrogenase maturation factors. To better understand the mechanism of GTP hydrolysis-dependent Ni delivery accomplished by HypB and HypA, our work focuses on the structure-function study of AfHypB from Archaeoglobus fulgidus and the functional interaction between AfHypA and AfHypB. / The intrinsic GTPase activity of AfHypB is very low, suggesting that AfHypB requires a G-protein activating protein (GAP) to activate its GTPase activity. Although AfHypB can interact with AfHypA to form 1:1 heterodimer, our data suggests that AfHypA is not a GAP for AfHypB. In addition, the FRET results showed that AfHypA could serve as a GEF (G-protein exchange factor) to activate the AfHypB from GDP-bound form to GTP-bound form and facilitate the dissociation of AfHypB dimer in the presence of GMPPNP. / Up to now, we have solved the structure of apo-AfHypB by X-ray crystallography. Crystals of AfHypB were grown using the hanging-drop-vapor-diffusion method and diffracted to ∼2.3 A. It belonged to space group P2(1)2(1)2, with unit cell dimensions a=72.49, b=82.33, c=68.66 A, alpha=beta=gamma=90°. Two molecules of AfHypB were found in an asymmetric unit. Structural comparison between the apo-AfHypB and GTP-bound HypB from M jannachii showed that the GTP binding broke the salt-bridge between K43 and D66, and induced conformational changes in the switch I loop and helix-3, which caused the HypB to form dimer and bind an extra Ni ion. The GTP-bound form of HypB was ready for Ni presenting. And GTP hydrolysis could induce the conformational changes of HypB in the switch I loop and helix-3, which dissociate the HypB dimer into the monomeric GDP-bound form. / Li, Ting. / Adviser: K. B. Wong. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 98-104). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.