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• W1991923195 abstract "Activation of a heterotrimeric G-protein by an agonist-stimulated G-protein-coupled receptor requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide binding pocket of the G-protein. To probe the molecular basis of this signaling process, we are applying high resolution NMR to track structural changes in an isotope-labeled, full-length G-protein α-subunit (Gα) chimera (ChiT) associated with G-protein βγ-subunit (Gβγ) and activated receptor (R*) interactions. Here, we show that ChiT can be functionally reconstituted with Gβγ as assessed by aluminum fluoride-dependent changes in intrinsic tryptophan fluorescence and light-activated rhodopsin-catalyzed guanine nucleotide exchange. We further show that 15N-ChiT can be titrated with Gβγ to form stable heterotrimers at NMR concentrations. To assess structural changes in ChiT upon heterotrimer formation, HSQC spectra of the 15N-ChiT-reconstituted heterotrimer have been acquired and compared with spectra obtained for GDP/Mg2+-bound 15N-ChiT in the presence and absence of aluminum fluoride and guanosine 5′-3-O-(thio)triphosphate (GTPγS)/Mg2+-bound 15N-ChiT. As anticipated, Gβγ association with 15N-ChiT results in 1HN, 15N chemical shift changes relative to the GDP/Mg2+-bound state. Strikingly, however, most 1HN, 15N chemical shift changes associated with heterotrimer formation are the same as those observed upon formation of the GDP⋅AlF4−/Mg2+- and GTPγS/Mg2+-bound states. Based on these comparative analyses, assembly of the heterotrimer appears to induce structural changes in the switch II and carboxyl-terminal regions of Gα (“preactivation”) that may facilitate the interaction with R* and subsequent GDP/GTP exchange. Activation of a heterotrimeric G-protein by an agonist-stimulated G-protein-coupled receptor requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide binding pocket of the G-protein. To probe the molecular basis of this signaling process, we are applying high resolution NMR to track structural changes in an isotope-labeled, full-length G-protein α-subunit (Gα) chimera (ChiT) associated with G-protein βγ-subunit (Gβγ) and activated receptor (R*) interactions. Here, we show that ChiT can be functionally reconstituted with Gβγ as assessed by aluminum fluoride-dependent changes in intrinsic tryptophan fluorescence and light-activated rhodopsin-catalyzed guanine nucleotide exchange. We further show that 15N-ChiT can be titrated with Gβγ to form stable heterotrimers at NMR concentrations. To assess structural changes in ChiT upon heterotrimer formation, HSQC spectra of the 15N-ChiT-reconstituted heterotrimer have been acquired and compared with spectra obtained for GDP/Mg2+-bound 15N-ChiT in the presence and absence of aluminum fluoride and guanosine 5′-3-O-(thio)triphosphate (GTPγS)/Mg2+-bound 15N-ChiT. As anticipated, Gβγ association with 15N-ChiT results in 1HN, 15N chemical shift changes relative to the GDP/Mg2+-bound state. Strikingly, however, most 1HN, 15N chemical shift changes associated with heterotrimer formation are the same as those observed upon formation of the GDP⋅AlF4−/Mg2+- and GTPγS/Mg2+-bound states. Based on these comparative analyses, assembly of the heterotrimer appears to induce structural changes in the switch II and carboxyl-terminal regions of Gα (“preactivation”) that may facilitate the interaction with R* and subsequent GDP/GTP exchange. Heterotrimeric G-proteins are intracellular signaling partners for the large family of seven transmembrane helix G-protein-coupled receptors (GPCRs). 3The abbreviations used are: GPCR, G-protein-coupled receptor; R*, the agonist-activated form of a GPCR; Gt, transducin; Gtα, the α-subunit of transducin; Gtβγ, the βγ-subunits of transducin; Gi1α, α-subunit of the inhibitory G-protein; GTPγS, guanosine 5′-O-3-thiotriphosphate; AlF4−, aluminum fluoride; ChiT, prodomain-released chimeric Gα; ROS, rod outer segment; HSQC, heteronuclear single quantum correlation; GEF, guanine nucleotide exchange factor; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid. 3The abbreviations used are: GPCR, G-protein-coupled receptor; R*, the agonist-activated form of a GPCR; Gt, transducin; Gtα, the α-subunit of transducin; Gtβγ, the βγ-subunits of transducin; Gi1α, α-subunit of the inhibitory G-protein; GTPγS, guanosine 5′-O-3-thiotriphosphate; AlF4−, aluminum fluoride; ChiT, prodomain-released chimeric Gα; ROS, rod outer segment; HSQC, heteronuclear single quantum correlation; GEF, guanine nucleotide exchange factor; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid. An agonist-activated GPCR (R*) binds to the inactive Gα(GDP)·Gβγ heterotrimer to promote GDP release and GTP uptake. The binding of GTP results in conformational changes in Gα (reviewed in Ref. 1Cabrera-Vera T.M. Vanhauwe J. Thomas T.O. Medkova M. Preininger A. Mazzoni M.R. Hamm H.E. Endocr. Rev. 2003; 24: 765-781Crossref PubMed Scopus (506) Google Scholar), ultimately leading to the dissociation of Gβγ. Both GTP-bound Gα and Gβγ are capable of initiating signaling cascades through interactions with downstream effectors such as adenylyl cyclase, cGMP phosphodiesterase, and various phospholipases or ion channels (reviewed in Ref. 2Preininger A.M. Hamm H.E. Sci. STKE. 2004; : RE3PubMed Google Scholar). The intrinsic GTPase activity of Gα results in the hydrolysis of GTP to GDP, returning Gα to its inactive GDP-bound ground state. Gα(GDP) eventually reassociates with Gβγ, thereby terminating all effector interactions. Activation of the retinal heterotrimeric G-protein transducin (Gt)by the GPCR rhodopsin represents an excellent model system to probe the molecular details of activated GPCR/G-protein interactions. Rhodopsin, the rod cell photoreceptor involved in dim light vision, is by far the best studied GPCR in terms of structure and function (3Ridge K.D. Abdulaev N.G. Sousa M. Palczewski K. Trends Biochem. Sci. 2003; 28: 479-487Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Light triggers the cis → trans isomerization of the retinal chromophore to initiate structural changes in the transmembrane helices that results in the formation of the light-activated signaling state, metarhodopsin II or R*. This is accompanied by small, yet functionally significant changes in the solvent-exposed cytoplasmic loops that lead to the formation of binding and activation sites for several signaling proteins, including Gt (2Preininger A.M. Hamm H.E. Sci. STKE. 2004; : RE3PubMed Google Scholar, 3Ridge K.D. Abdulaev N.G. Sousa M. Palczewski K. Trends Biochem. Sci. 2003; 28: 479-487Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 4Hubbell W.L. Altenbach C. Hubbell C.M. Khorana H.G. Adv. Protein Chem. 2003; 63: 243-290Crossref PubMed Scopus (339) Google Scholar). Importantly, crystal structures of the dark (inactive) state of bovine rhodopsin have been solved and refined providing valuable insights into the overall structural organization of this and other GPCRs (5Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4970) Google Scholar, 6Teller D.C. Okada T. Behnke C.A. Palczewski K. Stenkamp R.E. Biochemistry. 2001; 40: 7761-7772Crossref PubMed Scopus (625) Google Scholar, 7Okada T. Fujiyoshi Y. Silow M. Navarro J. Landau E.M. Schichida Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5982-5987Crossref PubMed Scopus (648) Google Scholar, 8Okada T. Sugihara M. Bondar A-N. Elstner M Entel P Buss V. J. Mol. Biol. 2004; 342: 571-583Crossref PubMed Scopus (924) Google Scholar, 9Li J. Edwards P.C. Burghammer M. Villa C. Schertler G.F.X. J. Mol. Biol. 2004; 343: 1409-1438Crossref PubMed Scopus (668) Google Scholar). Crystal structures of Gα, including Gtα (10Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 269: 1405-1412Crossref Scopus (745) Google Scholar, 11Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (519) Google Scholar, 12Mixon M.B. Lee E. Coleman D.E. Berghuis A.M. Gilman A.G. Sprang S.R. Science. 1995; 270: 954-960Crossref PubMed Scopus (266) Google Scholar, 13Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (698) Google Scholar, 14Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (528) Google Scholar, 15Sunahara R.K. Tesmer J.J. Gilman A.G. Sprang S.R. Science. 1997; 278: 1943-1947Crossref PubMed Scopus (262) Google Scholar) and Gβγ (16Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Crossref PubMed Scopus (705) Google Scholar) subunits, as well as Gαβγ heterotrimeric complexes (17Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1042) Google Scholar, 18Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1002) Google Scholar) have provided important insights into the structural rearrangements accompanying guanine nucleotide exchange and the GTPase cycle. The Gα subunit (Fig. 1) is composed of two domains: a guanine nucleotide binding domain with high structural homology to the Ras family of GTPases and an all α-helical domain that, in combination with the GTPase domain, helps to form a deep pocket for binding the guanine nucleotide (reviewed in Ref. 3Ridge K.D. Abdulaev N.G. Sousa M. Palczewski K. Trends Biochem. Sci. 2003; 28: 479-487Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Gα subunits contain three flexible regions designated switch I, switch II, and switch III that have been shown to adopt different conformations in the presence of GDP, GTPγS, and other nucleotide adducts and analogs (11Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (519) Google Scholar, 13Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (698) Google Scholar). The GTP-bound form of Gα, which can be mimicked by the nonhydrolyzable GTP analog GTPγS, has decreased affinity for Gβγ and increased affinity for Gα effectors. The GTPase domain of Gα (Fig. 1), a variation on the nucleotide-binding fold (19Rao S.T. Rossmann M.G. J. Mol. Biol. 1973; 76: 241-256Crossref PubMed Scopus (704) Google Scholar), adopts a similar conformation to that observed in crystal structures of elongation factor Tu and Ras (20La Cour T.F. Nyborg J. Thirup S. Clark B.F. EMBO J. 1985; 4: 2385-2388Crossref PubMed Scopus (275) Google Scholar, 21Tong L. de Vos A.M. Milburn M.V. Kim S.H. J. Mol. Biol. 1991; 217: 503-516Crossref PubMed Scopus (216) Google Scholar). The helical domain represents an insertion between the α1 helix and the β2 strand of the core GTPase domain and folds into a six-α-helix bundle. Interactions among residues that span the two-domain interface are thought to be involved in R*-catalyzed guanine nucleotide exchange and subsequent G-protein subunit dissociation (22Grishina G. Berlot C.H. J. Biol. Chem. 1998; 273: 15053-15060Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Gα subunits also contain an extended amino-terminal region of 26-36 amino acid residues. The first 23 residues appear to be disordered in the structure of Gtα in both the GDP- and the GTPγS-bound states (11Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (519) Google Scholar, 13Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (698) Google Scholar). In contrast, structures of the Gα(GDP)·Gβγ heterotrimer indicate that this region forms an α-helix that interacts with Gβ (17Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1042) Google Scholar, 18Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1002) Google Scholar). Recent findings suggest that in addition to Gβγ binding, N-myristoylation of Gα also imparts structural rigidity to the amino terminus (23Medkova M. Preininger A.M. Yu N.J. Hubbell W.L. Hamm H.E. Biochemistry. 2002; 41: 9962-9972Crossref PubMed Scopus (55) Google Scholar, 24Preininger A.M. Van Eps N. Yu N.J. Medkova M. Hubbell W.L. Hamm H.E. Biochemistry. 2003; 42: 7931-7941Crossref PubMed Scopus (34) Google Scholar). The extreme carboxyl-terminal region is also unresolved in the various crystal structures of Gtα (11Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (519) Google Scholar, 13Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (698) Google Scholar, 14Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (528) Google Scholar, 17Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1042) Google Scholar) and in the corresponding structures for the α-subunit of the inhibitory G-protein, Gi1α (10Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 269: 1405-1412Crossref Scopus (745) Google Scholar, 12Mixon M.B. Lee E. Coleman D.E. Berghuis A.M. Gilman A.G. Sprang S.R. Science. 1995; 270: 954-960Crossref PubMed Scopus (266) Google Scholar, 18Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1002) Google Scholar). High resolution NMR methods, however, have provided insights into the structures of carboxyl-terminal Gtα peptides encompassing this region when bound to light-activated rhodopsin (25Dratz E.A. Furstenau J.E. Lambert C.G. Thireault D.L. Rarick H. Schepers T. Pakhlevaniants S. Hamm H.E. Nature. 1993; 363: 276-281Crossref PubMed Scopus (151) Google Scholar, 26Kisselev O.G. Kao J. Ponder J.W. Fann Y.C. Gautam N. Marshall G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4270-4275Crossref PubMed Scopus (163) Google Scholar, 27Koenig B.W. Mitchell D.C. Konig S. Grzesiek S. Litman B.J. Bax A. J. Biomol. NMR. 2000; 16: 121-125Crossref PubMed Scopus (51) Google Scholar). Gβγ is a functional heterodimer that forms a stable structural unit. All Gβ subunits contain seven WD (Trp-Asp) repeats that form small anti-parallel β strands (28Jawad Z. Paoli M. Structure. 2002; 10: 447-454Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Crystal structures of the Gβγ dimer and Gαβγ heterotrimers show that the WD repeats fold into a four-stranded seven-bladed β-propeller, or toroid-like structure, whereas the amino terminus forms an extended α-helix (Fig. 1) (16Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Crossref PubMed Scopus (705) Google Scholar, 17Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1042) Google Scholar, 18Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1002) Google Scholar). Gγ folds into two α-helices (Fig. 1). The amino-terminal helix forms a coiled-coil with the α-helix of Gβ, whereas the carboxyl-terminal helix makes extensive contacts with the base of the Gβ propeller. Unlike Gα, the Gβγ dimer does not appear to change conformation upon dissociation from the heterotrimer (16Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Crossref PubMed Scopus (705) Google Scholar). Further, Gβγ association with Gα prevents Gβγ from activating its effectors (1Cabrera-Vera T.M. Vanhauwe J. Thomas T.O. Medkova M. Preininger A. Mazzoni M.R. Hamm H.E. Endocr. Rev. 2003; 24: 765-781Crossref PubMed Scopus (506) Google Scholar). Despite this wealth of available structural data, many questions remain surrounding R*/G-protein interactions and, specifically, the functional role of Gβγ in facilitating Gα(GDP) binding to the activated receptor and the subsequent release and exchange of bound GDP for GTP. Previous work has demonstrated that G-protein binding to an activated GPCR requires the presence of both Gα and Gβγ subunits, suggesting both a direct and indirect role for Gβγ in enabling R* interactions (33Herrmann R. Heck M. Henklein P. Henklein P. Kleuss C. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2004; 279: 24283-24290Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In addition, R*-catalyzed GDP release from Gα has been proposed to be mediated by Gβγ through two different mechanisms (the “lever arm” (59Iiri T. Farfel Z. Bourne H.R. Nature. 1998; 394: 35-38Crossref PubMed Scopus (162) Google Scholar) and “gear shift” (60Cherfils J. Chabre M. Trends Biochem. Sci. 2003; 28: 13-17Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) models) based on available structural and biochemical data, as well as analogies to proposed mechanisms for guanine exchange factor (GEF)-stimulated guanine nucleotide exchange in monomeric G-proteins. Clearly, a structural understanding of the conformational changes accompanying these processes poses many unique challenges given the inherently dynamic nature of the interactions. Thus, whereas the crystallographic studies have been instrumental in obtaining “snapshot” structures of dark state rhodopsin and have revealed guanine nucleotide-dependent structural rearrangements in the switch-I, -II, and -III regions of Gα, conformational changes in Gα that accompany R*/Gαβγ interactions remain unknown. Moreover, elucidation of the structural mechanism for R*-stimulated guanine nucleotide is severely hampered by a limited knowledge of the structures of the amino- and carboxyl-terminal regions of Gα, which are critical for a functional interaction with R*. As an initial step toward applying high resolution NMR methods to study the structure and dynamics of Gα in the heterotrimer state and in R*·Gαβγ complexes, we previously showed that a Gα chimera composed of amino acids 1-215 and 295-350 from Gtα, with an intervening sequence from Gi1α (Chi6) (29Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), can be expressed to high levels in a soluble form using a subtilisin prodomain fusion construct (proR8FKAM) and purified in a single step using an immobilized “slow cleaving” mutant form of subtilisin (30Abdulaev N.G. Zhang C. Dinh A. Ngo T. Bryan P.N. Brabazon D.M. Marino J.P. Ridge K.D. J. Biomol. NMR. 2005; 32: 31-40Crossref PubMed Scopus (18) Google Scholar). The mature, full-length chimera protein, which we call ChiT, exhibits a molecular mass of ∼40 kDa and has biochemical properties similar to those of Gtα isolated from bovine retina. Most importantly, milligram quantities of isotope-labeled ChiT can be purified using this expression/purification approach, enabling functional studies under solution NMR experimental conditions. In this paper, we show that ChiT can be reconstituted with Gtβγ subunits to form a functional heterotrimer that is amenable to structural analysis by high resolution NMR, thereby allowing us to probe differences in the conformation of Gα in various states as well as the structural basis for signal transfer from R* to the heterotrimeric G-protein. Surprisingly, analysis of the chemical shift patterns in the heteronuclear single quantum correlation (HSQC) spectrum of the 15N-ChiT-reconstituted heterotrimer are found to be very similar to the spectra acquired for the GDP⋅AlF4−/Mg2+- and GTPγS/Mg2+-bound states of ChiT. In particular, chemical shifts for resonances associated with residues that report on changes involving switch II, α3, and the carboxyl terminus are found to respond similarly to heterotrimer reconstitution and formation of the “transition/activated” states (i.e. GDP⋅AlF4−/Mg2+- and GTPγS/Mg2+-bound forms of ChiT). The functional implications of these observations with respect to the role of G-protein βγ-subunits in “preactivating” Gα for interaction with R* and GDP/GTP exchange are discussed. Materials—Complete™ EDTA-free protease inhibitor tablets and GTPγS were from Roche Applied Science, and [35S]GTPγS was from PerkinElmer Life Sciences. Phenylmethylsulfonyl fluoride, isopropyl-β-d-thiogalactopyranoside, GDP, and D2O were from Sigma, and blue Sepharose CL-6B was from Amersham Biosciences. 15NH4Cl and d11-Tris were from Spectra Stable Isotopes. The QuikChange II site-directed mutagenesis kit was from Stratagene, and Cymal-5 was from Anatrace. Bovine retinas were from W. Lawson Co. (Lincoln, NE). The pG58 expression vector, a fusion vector encoding a modified 77-amino acid prodomain region of subtilisin BPN′ (proR8FKAM), the pG58-derived expression vector encoding a Gtα/Gi1α chimera (Chi6) as a proR8FKAM fusion, and preparation of the S189 subtilisin BPN′ HiTrap NHS column have been described (30Abdulaev N.G. Zhang C. Dinh A. Ngo T. Bryan P.N. Brabazon D.M. Marino J.P. Ridge K.D. J. Biomol. NMR. 2005; 32: 31-40Crossref PubMed Scopus (18) Google Scholar, 31Ruan B. Fisher K.E. Alexander P.A. Doroshko V. Bryan P.N. Biochemistry. 2004; 43: 14539-14546Crossref PubMed Scopus (56) Google Scholar). Construction of ChiT Mutants—The F350A and F350W mutants were generated using the QuikChange II mutagenesis kit according to the manufacturer's instructions with the pG58 expression vector encoding the prodomain/Chi6 fusion serving as the template. The resulting mutations were verified by DNA sequencing. Construction of the W127F, W207F, and W254F mutants has been described (30Abdulaev N.G. Zhang C. Dinh A. Ngo T. Bryan P.N. Brabazon D.M. Marino J.P. Ridge K.D. J. Biomol. NMR. 2005; 32: 31-40Crossref PubMed Scopus (18) Google Scholar). Expression and Purification of Subtilisin Prodomain/Chi6 Fusions—Detailed protocols for the inducible expression and purification of isotope-labeled Gα have been described (30Abdulaev N.G. Zhang C. Dinh A. Ngo T. Bryan P.N. Brabazon D.M. Marino J.P. Ridge K.D. J. Biomol. NMR. 2005; 32: 31-40Crossref PubMed Scopus (18) Google Scholar). Briefly, Escherichia coli BL21 cells harboring the pG58 expression vector encoding the proR8FKAM fusion upstream of a chimeric Gα gene, Chi6 (29Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), were grown in minimal medium supplemented with 1 g/liter 15NH4Cl as the sole nitrogen source and 100 μg/ml ampicillin at 26 °C to A550 ∼ 0.3 and then induced with 30 μm isopropyl-β-d-thiogalactopyranoside for 12 h at 26 °C. The cell pellet was resuspended in 50 mm Tris-HCl, pH 8.0, containing 50 mm NaCl, 150 μm GDP, 5 mm MgCl2, 5 mm β-mercaptoethanol, 0.1 mm phenylmethylsulfonyl fluoride, and a protease inhibitor tablet and then disrupted by sonication. The supernatant obtained by centrifugation of the cell lysate at 100,000 × g for 45 min was collected and loaded onto a S189 subtilisin BPN′ HiTrap NHS column (31Ruan B. Fisher K.E. Alexander P.A. Doroshko V. Bryan P.N. Biochemistry. 2004; 43: 14539-14546Crossref PubMed Scopus (56) Google Scholar). The prodomain-released ChiT was eluted after 12 h in 10 mm Tris-HCl, pH 7.5, containing 100 mm NaCl, 5 mm MgCl2, 2.5 mm DTT, 50 μm GDP, and 0.1 mm phenylmethylsulfonyl fluoride (Buffer A). For NMR analysis, the purified, isotope-labeled protein was concentrated and dialyzed against 25 mm d11-Tris-HCl, pH 7.5, containing 100 mm NaCl, 5 mm magnesium acetate, 2.5 mm DTT, 50 μm GDP, and 5% glycerol (Buffer B), or eluted directly from the column in Buffer B. All ChiT constructs were purified in a similar manner. Reconstitution of ChiT with Gtβγ to Form the Gαβγ Heterotrimer—Gt was prepared from bovine retina by the method of Fung et al. (32Fung B.K.K. Hurley J.B. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 152-156Crossref PubMed Scopus (507) Google Scholar). To separate the Gtα and Gtβγ subunits, purified Gt in Buffer A containing 50% glycerol was diluted three times with the same buffer without glycerol and applied to blue Sepharose CL-6B equilibrated with 10 mm MOPS, pH 7.5, containing 5 mm magnesium acetate and 2.5 mm DTT. Gtβγ does not bind to the resin and is obtained from the flow-through. The column was washed with equilibration buffer, and Gtα eluted in the same buffer containing 2 m NaCl. The purified Gtα and Gtβγ subunits were concentrated and stored at -20 °C in Buffer A containing 50% glycerol (plus 50 μm GDP in the case of Gtα). The G-protein heterotrimer was reconstituted from ChiT and Gtβγ essentially as described (33Herrmann R. Heck M. Henklein P. Henklein P. Kleuss C. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2004; 279: 24283-24290Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Briefly, equimolar amounts of purified ChiT (or Gtα) were incubated with Gtβγ at room temperature for 15 min in 100 mm HEPES, pH 8.0, containing 1 mm EDTA, 10 mm MgSO4, 10 mm DTT, 10% glycerol, and 50 μm GDP. Heterotrimer formation was verified by analyzing an ali-quot of the mixture by gel filtration chromatography and then isolated in large quantities by preparative gel filtration chromatography. Prior to NMR experiments, the 15N-ChiT-reconstituted heterotrimer was concentrated and dialyzed against Buffer B. Fluorescence Assay for Measuring AlF4− -dependent Changes in Gtα, ChiT, ChiT-reconstituted Heterotrimer, and Gt—The tryptophan fluorescence of the GDP/Mg2+-bound form of ChiT and Gtα in isolation and in heterotrimer were determined in signal/reference mode essentially as described (34Phillips W.J. Cerione R.A. J. Biol. Chem. 1988; 263: 15498-15505Abstract Full Text PDF PubMed Google Scholar) using a FluoroMax-2 spectrofluorometer (Instruments SA, Edison, NJ) with a 0.3-cm square cuvette at 20 °C. Emission spectra were recorded over the wavelength range of 310-450 nm with an excitation wavelength of 290 nm. The spectral excitation and emission band pass was 5 nm for all spectra, with a signal integration time of 1 s. The 150-μl assay mixture contained a 150 nm concentration of either ChiT, Gtα, ChiT-reconstituted heterotrimer, or Gt in 50 mm Tris-HCl, pH 8.0, containing 50 mm NaCl, 2 mm MgCl2, and 1 mm DTT. The aluminum fluoride-induced changes were measured by adding 5-10 μl of AlCl3 (300 μm) and NaF (10 mm) separately or as a premixed solution. The fluorescence data were analyzed as previously described (35Skiba N. Thomas T.O. Hamm H.E. Methods Enzymol. 2000; 315: 502-524Crossref PubMed Google Scholar). Detergent Solubilization and Purification of Rhodopsin—Rhodopsin containing rod outer segments (ROS) were prepared from bovine retina using a standard protocol (36Wilden U. Kühn H. Biochemistry. 1982; 21: 3014-3022Crossref PubMed Scopus (267) Google Scholar). Rhodopsin was solubilized in phosphate-buffered saline, pH 7.0, containing 1% Cymal-5 and purified on immobilized rho-1D4 using methods previously described (37Oprian D.D. Molday R.S. Kaufman R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (381) Google Scholar, 38Ridge K.D. Lu Z. Liu X. Khorana H.G. Biochemistry. 1995; 34: 3261-3267Crossref PubMed Scopus (70) Google Scholar) in phosphate-buffered saline, pH 7.0, containing 0.1% Cymal-5. Rhodopsin concentrations were determined by UV-visible absorption spectroscopy using a λ6 spectrophotometer. Filter Binding Assay for Measuring G-protein-mediated Guanine Nucleotide Exchange—The functionality of the ChiT-reconstituted heterotrimer was examined by following the light-activated rhodopsincatalyzed uptake of [35S]GTPγS by G-protein using a nitrocellulose filter binding assay (39Abdulaev N.G. Ngo T. Chen R. Lu Z. Ridge K.D. J. Biol. Chem. 2000; 275: 39354-39363Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 40Wessling-Resnick M. Johnson G.L. J. Biol. Chem. 1987; 262: 3697-3705Abstract Full Text PDF PubMed Google Scholar). The filter-binding assay is based on the property that G-protein and its bound [35S]GTPγS are retained on nitrocellulose filters, whereas free [35S]GTPγS passes through the filters. The 500-μl assay mixtures initially contained 100 nm ROS rhodopsin solubilized and purified in Cymal-5 detergent and 4 μm Gt, Gtα reconstituted heterotrimer, or ChiT reconstituted heterotrimer in 10 mm Tris-HCl, pH 7.5, containing 100 mm NaCl, 5 mm MgCl2, and 2 mm DTT. The samples were illuminated (>495 nm) for 1 min or allowed to remain in darkness, and the reactions were initiated by the addition of 5 μm [35S]GTPγS. The final concentrations of Cymal-5 detergent and glycerol in the assay mixture were 0.08 and 5%, respectively. After various time intervals (30 s to 5 min) at 20 °C, 50 μl of the reaction mixture was removed and filtered through nitrocellulose with the aid of a vacuum manifold. The filters were washed four times with 5 ml of 10 mm Tris-HCl, pH 7.5, containing 100 mm NaCl, 5 mm MgCl2, and 2 mm DTT, dried, and analyzed for 35S radioactivity by scintillation counting. NMR Spectroscopy of 15N-ChiT and 15N-ChiT-reconstituted Heterotrimer—One-dimensional 15N-filtered and 15N HSQC water flip-back, water gate experiments (41Grzesiek S. Bax A. J. Am. Chem. Soc. 1993; 115: 12593-12594Crossref Scopus (1000) Google Scholar) were recorded at 30 °C on a Bruker AVANCE 600-MHz spectrometer (Bruker Instruments, Billerica, MA) equipped with a triple resonance 1H,13C,15N z axis gradient cryoprobe and linear amplifiers on all three channels. Spectra were collected on 15N-ChiT samples (150-300 μm) in Buffer B. The nitrogen frequency was centered at 118 ppm, and the proton frequency was centered on H2O (∼7.5 ppm). One-dimensional spectra were collected using a sweep width of 7,200" @default.
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• W1991923195 title "Heterotrimeric G-protein α-Subunit Adopts a “Preactivated” Conformation When Associated with βγ-Subunits" @default.
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