Book ChapterDOI
Expression of G-protein alpha subunits in Escherichia coli.
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TLDR
The introduction of a tobacco etch virus (TEV) polyprotein cleavage site between the hexahistidine tag and the G-protein α subunit permits the efficient removal of the tag by recombinant TEV protease.Abstract:
Publisher Summary This chapter describes a general method for expressing several G-protein α subunits in Escherichia coli (E. coli) at levels 10-100 times higher than achieved previously. G proteins are a family of guanine nucleotide-binding regulatory proteins that link a large number of cell surface receptors to regulation of several intracellular effectors. The chapter describes a method for purification of the recombinant proteins by affinity chromatography on a resin containing chelated Ni2+ after addition of an amino-terminal hexahistidine tag to the recombinant protein. Such purification is rapid and results in the isolation of highly purified protein in a single step. Furthermore, the introduction of a tobacco etch virus (TEV) polyprotein cleavage site between the hexahistidine tag and the G-protein α subunit permits the efficient removal of the tag by recombinant TEV protease.read more
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Journal ArticleDOI
The structure of the G protein heterotrimer Giα1β1γ2
Mark A. Wall,David E. Coleman,Ethan Lee,Jorge A. Iñiguez-Lluhi,Bruce A. Posner,Alfred G. Gilman,Stephen R. Sprang +6 more
TL;DR: The structure of the G protein heterotrimer Gi alpha 1(GDP)beta 1 gamma 2 (at 2.3 A) reveals two nonoverlapping regions of contact between alpha and beta, an extended interface between beta and nearly all of gamma, and limited interaction of alpha with gamma as mentioned in this paper.
Journal ArticleDOI
Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis.
David E. Coleman,Albert M. Berghuis,Ethan Lee,Maurine E. Linder,Alfred G. Gilman,Stephen R. Sprang +5 more
TL;DR: AlF4- complexes formed by the G protein Gi alpha 1 demonstrate specific roles in transition-state stabilization for two highly conserved residues, suggesting a mechanism that may promote release of the beta gamma subunit complex when the alpha subunit is activated by GTP.
Journal ArticleDOI
Structure of RGS4 Bound to AlF4−-Activated Giα1: Stabilization of the Transition State for GTP Hydrolysis
TL;DR: RGS4 appears to catalyze rapid hydrolysis of GTP primarily by stabilizing the switch regions of G(i alpha1), although the conserved Asn-128 from RGS4 could also play a catalytic role by interacting with the hydrolytic water molecule or the side chain of Gln-204.
Journal ArticleDOI
GAIP and RGS4 Are GTPase-Activating Proteins for the Gi Subfamily of G Protein α Subunits
TL;DR: Two RGS family members, GAIP and RGS4, are GTPase-activating proteins (GAPs), accelerating the rate of GTP hydrolysis by Gi alpha 1 at least 40-fold, consistent with their proposed role as negative regulators of G protein-mediated signaling.
Journal ArticleDOI
Conformational biosensors reveal GPCR signalling from endosomes
Roshanak Irannejad,Jin Cui Tomshine,Jon R. Tomshine,Michael Chevalier,Jacob P. Mahoney,Jan Steyaert,Søren G. F. Rasmussen,Roger K. Sunahara,Hana El-Samad,Bo Huang,Mark von Zastrow +10 more
TL;DR: It is shown that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application, providing direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membranes.
References
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Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes
F W Studier,B A Moffatt +1 more
TL;DR: A gene expression system based on bacteriophage T7 RNA polymerase has been developed and high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and/or stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degradation.
Journal ArticleDOI
Diversity of G proteins in signal transduction.
TL;DR: The heterotrimeric guanine nucleotide-binding proteins acting as switches that regulate information processing circuits connecting cell surface receptors to a variety of effectors generate the pathways that modulate cellular responses to complex chemical signals.
Journal ArticleDOI
The guanine nucleotide activating site of the regulatory component of adenylate cyclase. Identification by ligand binding.
TL;DR: There is no reversal of activation or binding under activating conditions and the rate constant for activation is unchanged from 10 mi to 100 PM GTPyS, so reversal of binding and activation, which occurs only in the absence of divalent cation, is not a first order process.
Journal ArticleDOI
Characterization of transducin from bovine retinal rod outer segments. I. Separation and reconstitution of the subunits.
TL;DR: Findings strongly suggest that the T beta gamma subunit is an activator of the GTPase activity to enable the T alpha subunit to interact with rhodopsin effectively.
The Guanine Nucleotide Activating Site of the Regulatory Component of Adenylate Cyclase
TL;DR: In this article, the authors developed methods for the assessment of binding of radiolabeled guanosine-5'(3-0-thio)triphosphate (GTPyS) and guanine-6'(P,yimin0) triphosphates (Gpp(NH)p) to homogeneous preparations of adenylate cyclase (G/F).