FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2064118841> ?p ?o ?g. }

• W2064118841 endingPage "7648" @default.
• W2064118841 startingPage "7635" @default.
• W2064118841 abstract "Solution NMR studies of a 15N-labeled G-protein α-subunit (Gα) chimera (15N-ChiT)-reconstituted heterotrimer have shown previously that G-protein βγ-subunit (Gβγ) association induces a “pre-activated” conformation that likely facilitates interaction with the agonist-activated form of a G-protein-coupled receptor (R*) and guanine nucleotide exchange (Abdulaev, N. G., Ngo, T., Zhang, C., Dinh, A., Brabazon, D. M., Ridge, K. D., and Marino, J. P. (2005) J. Biol. Chem. 280, 38071-38080). Here we demonstrated that the 15N-ChiT-reconstituted heterotrimer can form functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin (R*), as well as a soluble mimic of R*. NMR methods were used to track R*-triggered guanine nucleotide exchange and release of guanosine 5′-O-3-thiotriphosphate (GTPγS)/Mg2+-bound ChiT. A heteronuclear single quantum correlation (HSQC) spectrum of R*-generated GTPγS/Mg2+-bound ChiT revealed 1HN, 15N chemical shift changes relative to GDP/Mg2+-bound ChiT that were similar, but not identical, to those observed for the GDP·AlF4-/Mg2+-bound state. Line widths observed for R*-generated GTPγS/Mg2+-bound 15N-ChiT, however, indicated that it is more conformationally dynamic relative to the GDP/Mg2+- and GDP·AlF4-/Mg2+-bound states. The increased dynamics appeared to be correlated with Gβγ and R* interactions because they are not observed for GTPγS/Mg2+-bound ChiT generated independently of R*. In contrast to R*, a soluble mimic that does not catalytically interact with G-protein (Abdulaev, N. G., Ngo, T., Chen, R., Lu, Z., and Ridge, K. D. (2000) J. Biol. Chem. 275, 39354-39363) is found to form a stable complex with the GTPγS/Mg2+-exchanged heterotrimer. The HSQC spectrum of 15N-ChiT in this complex displays a unique chemical shift pattern that nonetheless shares similarities with the heterotrimer and GTPγS/Mg2+-bound ChiT. Overall, these results demonstrated that R*-induced changes in Gα can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation. Solution NMR studies of a 15N-labeled G-protein α-subunit (Gα) chimera (15N-ChiT)-reconstituted heterotrimer have shown previously that G-protein βγ-subunit (Gβγ) association induces a “pre-activated” conformation that likely facilitates interaction with the agonist-activated form of a G-protein-coupled receptor (R*) and guanine nucleotide exchange (Abdulaev, N. G., Ngo, T., Zhang, C., Dinh, A., Brabazon, D. M., Ridge, K. D., and Marino, J. P. (2005) J. Biol. Chem. 280, 38071-38080). Here we demonstrated that the 15N-ChiT-reconstituted heterotrimer can form functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin (R*), as well as a soluble mimic of R*. NMR methods were used to track R*-triggered guanine nucleotide exchange and release of guanosine 5′-O-3-thiotriphosphate (GTPγS)/Mg2+-bound ChiT. A heteronuclear single quantum correlation (HSQC) spectrum of R*-generated GTPγS/Mg2+-bound ChiT revealed 1HN, 15N chemical shift changes relative to GDP/Mg2+-bound ChiT that were similar, but not identical, to those observed for the GDP·AlF4-/Mg2+-bound state. Line widths observed for R*-generated GTPγS/Mg2+-bound 15N-ChiT, however, indicated that it is more conformationally dynamic relative to the GDP/Mg2+- and GDP·AlF4-/Mg2+-bound states. The increased dynamics appeared to be correlated with Gβγ and R* interactions because they are not observed for GTPγS/Mg2+-bound ChiT generated independently of R*. In contrast to R*, a soluble mimic that does not catalytically interact with G-protein (Abdulaev, N. G., Ngo, T., Chen, R., Lu, Z., and Ridge, K. D. (2000) J. Biol. Chem. 275, 39354-39363) is found to form a stable complex with the GTPγS/Mg2+-exchanged heterotrimer. The HSQC spectrum of 15N-ChiT in this complex displays a unique chemical shift pattern that nonetheless shares similarities with the heterotrimer and GTPγS/Mg2+-bound ChiT. Overall, these results demonstrated that R*-induced changes in Gα can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation. G-protein coupled receptors (GPCRs) 3The abbreviations used are: GPCR, G-protein coupled receptor; TM, transmembrane; R*, the agonist activated form of a GPCR; GTPγS, guanosine 5′-O-3-thiotriphosphate; CMC, critical micelle concentration; DM, n-dodecyl β-d-maltopyranoside; ChiT, prodomain released chimeric Gα; ROS, rod outer segment; HSQC, heteronuclear single quantum correlation; HPTRX/CDEF, protein chimera containing segments from the CD and EF loops of bovine opsin grafted into a thioredoxin loop.3The abbreviations used are: GPCR, G-protein coupled receptor; TM, transmembrane; R*, the agonist activated form of a GPCR; GTPγS, guanosine 5′-O-3-thiotriphosphate; CMC, critical micelle concentration; DM, n-dodecyl β-d-maltopyranoside; ChiT, prodomain released chimeric Gα; ROS, rod outer segment; HSQC, heteronuclear single quantum correlation; HPTRX/CDEF, protein chimera containing segments from the CD and EF loops of bovine opsin grafted into a thioredoxin loop. represent a diverse group of seven transmembrane (TM) helix receptors that require agonist-dependent activation to initiate heterotrimeric (αβγ) G-protein-mediated intracellular signaling cascades. GPCR activation of cognate G-proteins are the first steps in cellular communication pathways responsible for signaling cascades that mediate vision, olfaction, taste, and the action of numerous hormones and neurotransmitters (1Lundstrom K. Trends Biotechnol. 2005; 23: 103-108Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Activation of a G-protein by its agonist-stimulated GPCR (R*) requires the propagation of structural signals from the receptor-binding interface to the guanine nucleotide-binding pocket. The structural basis for the interaction of a GPCR with its cognate G-protein and the subsequent activation of the G-protein by R* are not fully understood. Using signaling of the retinal G-protein transducin (Gt) by rhodopsin as a model system, we are applying solution NMR methods to track changes in the G-protein α-subunit (Gα) associated with activated R* interactions. Rhodopsin, the rod cell photoreceptor involved in dimlight vision, represents one of the best studied GPCRs in terms of structure and function (2Ridge K.D. Abdulaev N.G. Sousa M. Palczewski K. Trends Biochem. Sci. 2003; 28: 479-487Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 3Abdulaev N.G. Ridge K.D. Briggs W.R. Spudich J.L. Handbook of Photosensory Receptors. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany2005: 77-92Crossref Scopus (5) Google Scholar). Photon capture triggers cis → trans isomerization of the retinal chromophore, which initiates structural changes in the TM helices resulting in the formation of the light-activated signaling state metarhodopsin II, R*. This is accompanied by functionally significant changes at the cytoplasmic surface that leads to the formation of binding and activation sites for several signaling proteins, including Gt (4Kühn H. Mommertz O. Hargrave P.A. Biochim. Biophys. Acta. 1982; 679: 95-100Crossref Scopus (46) Google Scholar, 5Pellicone C. Nullans G. Cook N. Virmaux N. Biochem. Biophys. Res. Commun. 1985; 127: 816-821Crossref PubMed Scopus (15) Google Scholar, 6Farahbakhsh Z.T. Hideg K. Hubbell W.L. Science. 1993; 262: 1416-1419Crossref PubMed Scopus (147) Google Scholar, 7Farahbakhsh Z.T. Ridge K.D. Khorana H.G. Hubbell W.L. Biochemistry. 1995; 34: 8812-8819Crossref PubMed Scopus (186) Google Scholar, 8Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1111) Google Scholar, 9Abdulaev N.G. Ridge K.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12854-12859Crossref PubMed Scopus (77) Google Scholar, 10Hubbell W.L. Altenbach C. Hubbell C.M. Khorana H.G. Adv. Protein Chem. 2003; 63: 243-290Crossref PubMed Scopus (340) Google Scholar). Crystal structures for the inactive (dark) state of rhodopsin have provided a detailed view of the retinal binding site and the cytoplasmic region (11Palczewski 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 (5023) Google Scholar, 12Teller D.C. Okada T. Behnke C.A. Palczewski K. Stenkamp R.E. Biochemistry. 2001; 40: 7761-7772Crossref PubMed Scopus (628) Google Scholar, 13Okada 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 (656) Google Scholar, 14Okada T. Sugihara M. Bondar A.-N. Elstner M. Entel P. Buss V. J. Mol. Biol. 2004; 342: 571-583Crossref PubMed Scopus (939) Google Scholar, 15Li J. Edwards P.C. Burghammer M. Villa C. Schertler G.F.X. J. Mol. Biol. 2004; 343: 1409-1438Crossref PubMed Scopus (673) Google Scholar). Although remarkably informative, the crystal structures provide few solid insights into the mechanism of signal transfer from R* to Gt. Binding of heterotrimeric G-proteins to activated GPCRs requires the presence of both Gα and G-protein βγ-subunits (Gβγ). The following three regions on the α-subunit of Gt (Gtα) are known to be important for receptor interactions; the amino-terminal 23 residues, an internal sequence from amino acids 305-315, and the carboxyl-terminal 11 amino acids (16van Dop C. Yamanaka G. Steinberg F. Sekura R.D. Manclark C.R. Stryer L. Bourne H.R. J. Biol. Chem. 1984; 259: 23-26Abstract Full Text PDF PubMed Google Scholar, 17West Jr., R.E. Moss J. Vaughan M. Liu T. Liu T.Y. J. Biol. Chem. 1985; 260: 14428-14430Abstract Full Text PDF PubMed Google Scholar, 18Hamm H.E. Deretic D. Arendt A. Hargrave P.A. Koenig B. Hoffman K.P. Science. 1988; 241: 832-835Crossref PubMed Scopus (394) Google Scholar). Upon binding to R*, Gtα is thought to undergo structural changes in both the amino- and carboxyl-terminal regions. High resolution crystal structures of Gα subunits, including Gtα (19Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 269: 1405-1412Crossref Scopus (752) Google Scholar, 20Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (530) Google Scholar, 21Mixon M.B. Lee E. Coleman D.E. Berghuis A.M. Gilman A.G. Sprang S.R. Science. 1995; 270: 954-960Crossref PubMed Scopus (267) Google Scholar, 22Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (707) Google Scholar, 23Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (534) Google Scholar, 24Sunahara R.K. Tesmer J.J. Gilman A.G. Sprang S.R. Science. 1997; 278: 1943-1947Crossref PubMed Scopus (264) Google Scholar), Gβγ (25Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Crossref PubMed Scopus (707) Google Scholar), and Gαβγ heterotrimeric complexes (Refs. 26Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1047) Google Scholar and 27Wall 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 (1009) Google Scholar, Fig. 1A), have provided important insights into the structural rearrangements accompanying guanine nucleotide exchange and the GTPase cycle, particularly in the conformationally flexible switch regions. In most of the crystal structures, however, the residues at the extreme carboxyl terminus of Gα are disordered and/or not visible. This is consistent with findings from transferred nuclear Overhauser enhanced spectroscopy NMR studies (28Dratz 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 (152) Google Scholar, 29Kisselev 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, 30Koenig B.W. Mitchell D.C. Konig S. Grzesiek S. Litman B.J. Bax A. J. Biomol. NMR. 2000; 16: 121-125Crossref PubMed Scopus (52) Google Scholar) on 11 amino acid carboxyl-terminal peptides derived from Gtα (Gtα (340-350) peptides), which show that these peptides are largely unstructured in solution and in the presence of dark state rod outer segment (ROS) rhodopsin, but undergo significant structural changes upon binding to light-activated rhodopsin. Similarly, the helical structure of the amino terminus of Gα appears transient and is only ordered in crystal structures of the heterotrimer (26Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1047) Google Scholar, 27Wall 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 (1009) Google Scholar). Results from a study in which fluorescent and spin-label probes were introduced at specific positions in the amino-terminal region of Gα are consistent with the amino terminus by assuming an ordered helical conformation only in Gtαβγ (31Medkova M. Preininger A.M. Yu N.-J. Hubbell W.L. Hamm H.E. Biochemistry. 2002; 41: 9962-9972Crossref PubMed Scopus (56) Google Scholar). High resolution structural analysis of R*/G-protein interactions poses many significant challenges given the inherently dynamic nature of this process. The R*/Gt interaction can be viewed as taking place in at least five discrete biochemical reaction steps (Fig. 1B). These include R* binding to Gtαβγ·GDP to form the R*·Gtαβγ·GDP complex (steps 1 and 2), GDP dissociation from the R*·Gtαβγ·GDP complex to form an R*·Gtαβγ[empty] complex (step 3), GTP uptake by the R*·Gtαβγ[empty] complex to form R*·Gtαβγ·GTP (step 4), and dissociation of Gtαβγ·GTP from R* followed by Gtα·GTP from Gtβγ (step 5), with R* now free for interaction with another Gtαβγ·GDP (32Herrmann 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 (80) Google Scholar). Although the above mentioned crystallographic studies have been instrumental for obtaining static three-dimensional structures of dark state rhodopsin and various guanine nucleotide-bound states of Gα, and biochemical and mutational approaches have provided a wealth of information about the nature of R*/Gt interactions, a structural representation of the R*-Gt complex(es) remains poorly defined. Clearly, a comprehensive description of the structures involved in these reaction steps would provide important insights into the mechanisms governing activated GPCR/G-protein interactions. We have shown previously that a Gα chimera consisting of sequences from Gtα and Gi1α (Chi6; see Ref. 33Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) can be expressed to high levels in a soluble form by using a subtilisin prodomain (proR8FKAM) fusion construct and milligram quantities of prodomain-released, full-length, isotope-labeled Gα (ChiT) purified in a single step by using an immobilized “slow cleaving” mutant form of subtilisin (34Abdulaev 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). This has allowed us to pursue functional studies under NMR experimental conditions that provide insights into the solution structures of Gα in various states. We have also shown that isotope-labeled ChiT can be reconstituted with Gtβγ subunits to form a functional heterotrimer that is amenable to structural analysis by high resolution NMR (35Abdulaev N.G. Ngo T. Zhang C. Dinh A. Brabazon D.M. Ridge K.D. Marino J.P. J. Biol. Chem. 2005; 280: 38071-38080Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This latter work revealed that Gβγ binding to ChiT induces structural changes in the guanine nucleotide binding and carboxyl-terminal regions of ChiT, leading to a “preactivated” state that may facilitate interaction with R* and subsequent GDP/GTP exchange. Here we have now applied high resolution NMR to begin to probe the structural basis for the propagation of signals from R* to the G-protein, with the specific goal of developing more robust models for the structural changes in Gα that accompany the signal transfer process. Specifically, NMR methods have been used to track the complete cycle of guanine nucleotide exchange in 15N-ChiT-reconstituted heterotrimer that is triggered by light-activated rhodopsin (R*), thereby providing new insights into Gα conformational changes-associated signal propagation from an activated GPCR. Using similar NMR approaches, guanine nucleotide exchange in a 15N-ChiT-reconstituted heterotrimer stimulated by a soluble mimic of R* has been monitored. In contrast to R*, the soluble mimic remains bound to the nucleotide-exchanged heterotrimer forming a trapped, stable complex akin to the R*·Gtαβγ·GTP intermediate in the reaction pathway. Materials—Cyclohexylpentyl-β-d-maltoside (Cymal-5) and n-dodecyl β-d-maltopyranoside (DM) were from Anatrace. GTPγS was from Roche Applied Science, and Ni2+-nitrilotriacetic acid-agarose resin was from Qiagen. [35S]GTPγS was from PerkinElmer Life Sciences. Anti-Gtα and anti-Gtβ antibodies were from Affinity BioReagents, and the anti-rhodopsin antibody K42-41L (37Adamus G. Zam Z.S. Arendt A. Palczewski K. McDowell J.H. Hargrave P.A. Vision Res. 1991; 31: 17-31Crossref PubMed Scopus (169) Google Scholar) was a gift from Prof. Paul Hargrave (University of Florida). Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies were from Santa Cruz Biotechnology, and protein G-Sepharose was a gift from Prof. Philip Bryan (University of Maryland Biotechnology Institute). The pG58 expression vector, a fusion vector encoding a modified 77-amino acid prodomain region of subtilisin BPN′ (proR8FKAM), and the pG58-derived expression vector encoding a Gα chimera (Chi6) as a proR8FKAM fusion have been described (34Abdulaev 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, 36Ruan B. Fisher K.E. Alexander P.A. Doroshko V. Bryan P.N. Biochemistry. 2004; 43: 14539-14546Crossref PubMed Scopus (60) Google Scholar). The sources of other materials used in this investigation have been reported (34Abdulaev 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, 35Abdulaev N.G. Ngo T. Zhang C. Dinh A. Brabazon D.M. Ridge K.D. Marino J.P. J. Biol. Chem. 2005; 280: 38071-38080Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 38Abdulaev 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). Expression and Purification of Subtilisin Prodomain/Chi6 Fusions—Methods for the inducible bacterial expression and purification of isotope-labeled wild-type and mutant Gα using the proR8FKAM/Chi6 fusion and immobilized S189 subtilisin BPN′ have been described (34Abdulaev 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). To generate the GTPγS/Mg2+-bound form of ChiT independently of R*, GDP was omitted from the cell lysis and column purification buffers in order to obtain an `empty pocket' state of Gα that could be subsequently reconstituted with GTPγS. Prior to NMR analysis, the purified and isotope-labeled proteins were concentrated and dialyzed against 25 mm d11Tris-HCl, pH 7.5, containing 100 mm NaCl, 5 mm magnesium acetate, 2.5 mm dithiothreitol, and 5% glycerol (Buffer A). Expression and Purification of HPTRX/CDEF—Detailed protocols for the inducible expression and purification of HPTRX/CDEF, a soluble mimic of R*, have been described (38Abdulaev 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). Prior to NMR experiments, purified HPTRX/CDEF was concentrated and dialyzed against Buffer A. Detergent Solubilization and Purification of Rhodopsin—ROS rhodopsin from bovine retina was solubilized and purified in Cymal-5 or DM detergent on rho-1D4-Sepharose essentially as described (39Oprian D.D. Molday R.S. Kaufmann R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (386) Google Scholar, 40Ridge K.D. Lee S.S.J. Yao L.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3204-3208Crossref PubMed Scopus (113) Google Scholar). Rhodopsin concentrations were determined by UV-visible spectroscopy at 20 °C using a λ25 spectrophotometer (PerkinElmer Life Sciences). Prior to NMR experiments, rhodopsin preparations were concentrated and dialyzed against Buffer A containing 0.08% (w/v) Cymal-5. Filter Binding Assay for Measuring G-protein-mediated Guanine Nucleotide Exchange—The ability of detergent-solubilized rhodopsin preparations to catalyze the uptake of [35S]GTPγS by Gt was determined by initial rate analysis, and at equilibrium, using a nitrocellulose filter binding assay essentially as described (41Nakayama T.A. Khorana H.G. J. Biol. Chem. 1991; 266: 4269-4275Abstract Full Text PDF PubMed Google Scholar). For initial rate analyses, the reaction mixtures contained 6.7 nm Cymal-5- or DM-solubilized and -purified ROS rhodopsin and 5 μm [35S]GTPγS in 10 mm Tris-HCl, pH 7.5, containing 100 mm NaCl, 5 mm MgCl2, and 2.5 mm dithiothreitol (Buffer B). After illumination (>495 nm) for 1 min at 20 °C, the reactions were initiated by the addition of 4 μm Gt. The total reaction volume was 250 μl, and the final concentrations of Cymal-5, DM, and glycerol in the assay mixtures were 0.08, 0.015, and 5% (w/v), respectively. At various time intervals (5-20 s), a 50-μl aliquot was removed, rapidly filtered through nitrocellulose with the aid of a vacuum manifold, and the filters immediately washed three to four times with 5 ml of Buffer B to remove free, unbound [35S]GTPγS. The filters were dried, and the G-protein-bound [35S]GTPγS was determined by scintillation counting. For the equilibrium assays, the same reaction mixture containing 4 μm Gt was illuminated for 1 min, the reaction allowed to proceed for 2 h at 20 °C, and the extent of GDP/GTPγS exchange determined as described above. Identical reactions were performed in the dark for both the initial rate and equilibrium assays. The activity in the dark was subtracted from that in the light for determination of the kinetic and equilibrium values, which are reported as averages ± S.E. Rate of Metarhodopsin II (R*) Decay in the Absence and Presence of Gt—The rate of retinal release upon R* decay in 0.08% Cymal-5 or 0.015% DM, and in the presence Gt, was determined by following the decrease in protonated retinyl-Schiff base as measured at 440 nm after acidification essentially as described (42Farrens D.L. Khorana H.G. J. Biol. Chem. 1995; 270: 5073-5076Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 43Blazynski C. Ostroy S.E. Vision Res. 1981; 21: 833-841Crossref PubMed Scopus (11) Google Scholar, 44Blazynski C. Ostroy S.E. Vision Res. 1984; 24: 459-470Crossref PubMed Scopus (28) Google Scholar). Briefly, purified rhodopsin (1.17 μm) in 25 mm Tris-HCl, pH 7.5, containing 100 mm NaCl, 5 mm magnesium acetate, 2.5 mm dithiothreitol, 5% glycerol (Buffer C), and 0.08% Cymal-5 or 0.015% DM was illuminated (>495 nm for 1 min) at 20 °C in the absence or presence of Gt (1.4 μm, 1.2-fold excess over rhodopsin). Aliquots (400 μl) were removed at specific time points (typically at 1, 15, 30, 60, 120, 240, and 480 min) and acidified to pH ∼3 by addition of 2 n H2SO4. After mixing, a UV-visible spectrum (650 nm-250 nm) was recorded, and the amount of the protonated retinyl-Schiff base (440 nm) remaining as a function of time was determined. Reconstitution of ChiT with Gtβγ to Form the Gαβγ Heterotrimer—The G-protein heterotrimer was reconstituted from isotope-labeled ChiT and Gtβγ essentially as described (35Abdulaev N.G. Ngo T. Zhang C. Dinh A. Brabazon D.M. Ridge K.D. Marino J.P. J. Biol. Chem. 2005; 280: 38071-38080Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Prior to NMR experiments, heterotrimer preparations were concentrated and dialyzed against Buffer A or Buffer A containing 0.08% Cymal-5. Immunoprecipitation and Analysis of the Gαβγ-GTPγS·HPTRX/CDEF Complex—Equimolar concentrations of HPTRX/CDEF and ChiT-reconstituted heterotrimer (500 nm) in Buffer C were mixed with GTPγS (600 nm) and incubated for 30 min at 20 °C. An aliquot of anti-Gtβ antibody (20 μl of a 1 mg/ml solution) was added to the mixture, followed by 200 μl of pre-cleaned protein G-Sepharose. After gentle mixing for 30 min at 20 °C, the beads were allowed to settle, the supernatant removed, and the beads washed five times with 1 ml of Buffer C. The washed beads were resuspended in reducing SDS-PAGE sample buffer (250 μl), and aliquots (50 μl) were examined by reducing SDS-PAGE (45Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (207002) Google Scholar) using a 5% stacking and a 16% resolving gel. The immunoprecipitated proteins were electroblotted onto poly(vinyl difluoride) membranes (46Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar), detected with the K42-41L (HPTRX/CDEF), anti-Gtα (ChiT), and anti-Gtβ (Gtβ) antibodies, or a mixture of these primary antibodies, and horseradish peroxidase-conjugated goat anti-mouse and/or anti-rabbit antibodies. The proteins were visualized by chemiluminescence. NMR Spectroscopy—One-dimensional 1H- and 15N-filtered 1H water flip-back, water gate, 15N-decoupled spectra and two-dimensional 15N-HSQC water flip-back, water gate spectra (47Grzesiek S. Bax A. J. Am. Chem. Soc. 1993; 115: 12593-12594Crossref Scopus (1012) Google Scholar) were acquired at 30 °C using 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 uniformly 15N-labeled samples (15N-ChiT) dissolved in Buffer A at concentrations of 150-300 μm. The nitrogen frequency was centered at 118 ppm and the proton frequency on H2O (∼7.5 ppm). One-dimensional spectra were collected using a sweep width of 7,200 Hz and 2,048 complex points, and two-dimensional data were acquired using sweep widths of 7,200 Hz in ω2 and 2,000 Hz in ω1, 2,048 by 64 complex data points in t2 and t1, respectively, (t1(max) = 293 ms and t2(max) = 64 ms) and 128 scans per increment. NMR samples containing rhodopsin were placed in the spectrometer under dim-red light conditions. To initiate the guanine nucleotide exchange reaction, rhodopsin was illuminated with >495 nm light for 1 min prior to spectral acquisition. For NMR samples containing HPTRX/CDEF, the protein was added directly to the reaction mixture to stimulate guanine nucleotide exchange. All spectra were processed and analyzed on a SGI UNIX work station using NMRPipe (48Delaglio F. Grzesiek S. Vuister G. Zhu G. Pfeifer J. Bax A. J. Biolmol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11529) Google Scholar). Trp indole and Phe-350 amide 1NH and 15N resonances were assigned using ChiT mutants as described previously (34Abdulaev 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, 35Abdulaev N.G. Ngo T. Zhang C. Dinh A. Brabazon D.M. Ridge K.D. Marino J.P. J. Biol. Chem. 2005; 280: 38071-38080Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The aluminum fluoride (AlF4-) adduct of GDP/Mg2+-bound ChiT was formed by addition of NaF (10 mm) and AlCl3 (300 μm) directly to the NMR tube. Other Methods—A fluorescence assay for monitoring Gt activation by HPTRX/CDEF was performed essentially as described (38Abdulaev 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). Protein determinations were done as described previously (49Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7127) Google Scholar). Experimental Design and General Considerations—Having previously demonstrated that 15N-ChiT can be functionally expressed as a subtilisin BPN′ prodomain fusion and purified on immobilized S189 subtilisin BPN′, reconstituted with unlabeled Gtβγ subunits to form a heterotrimer (∼85 kDa), and characterized by high resolution NMR (34Abdulaev 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, 35Abdulaev N.G. Ngo T. Zhang C. Dinh A. Brabazon D.M. Ridge K.D. Marino J.P. J. Biol. Chem. 2005; 280: 38071-38080Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), it was of keen interest to now investigate whether NMR could also be used to follow some of the reaction steps involved in R*-catalyzed guanine nucleotide exchange (Fig. 1B). Because these solution NMR studies would necessitate the use of rhodopsin, an ∼40-kDa light-sensitive integral membrane protein, it was necessary to identify a detergent that could not only effectively solubilize rhodopsin but also support the formation of R* at concentrations below the critical micelle concentration (CMC). The rationale for choosing such a detergent for these NMR studies was as follows. At detergent concentrations below the CMC, an R*-G-protein complex would be expected to behave essent" @default.
• W2064118841 created "2016-06-24" @default.
• W2064118841 creator A5008721821 @default.
• W2064118841 creator A5057223751 @default.
• W2064118841 creator A5057631578 @default.
• W2064118841 creator A5059670809 @default.
• W2064118841 creator A5073143137 @default.
• W2064118841 creator A5075596275 @default.
• W2064118841 date "2006-03-01" @default.
• W2064118841 modified "2023-09-29" @default.
• W2064118841 title "Conformational Changes Associated with Receptor-stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-protein α-Subunit" @default.
• W2064118841 cites W1491721575 @default.
• W2064118841 cites W1503305141 @default.
• W2064118841 cites W1517746500 @default.
• W2064118841 cites W1521235731 @default.
• W2064118841 cites W1547901668 @default.
• W2064118841 cites W1566050966 @default.
• W2064118841 cites W1573946811 @default.
• W2064118841 cites W1593806256 @default.
• W2064118841 cites W1615152444 @default.
• W2064118841 cites W1628126976 @default.
• W2064118841 cites W1649341288 @default.
• W2064118841 cites W1736189198 @default.
• W2064118841 cites W1932052686 @default.
• W2064118841 cites W1942777601 @default.
• W2064118841 cites W1974770940 @default.
• W2064118841 cites W1977920448 @default.
• W2064118841 cites W1978798664 @default.
• W2064118841 cites W1980420481 @default.
• W2064118841 cites W1983831869 @default.
• W2064118841 cites W1986215328 @default.
• W2064118841 cites W1988819965 @default.
• W2064118841 cites W1990457307 @default.
• W2064118841 cites W1991923195 @default.
• W2064118841 cites W1993466966 @default.
• W2064118841 cites W1994019984 @default.
• W2064118841 cites W1997161001 @default.
• W2064118841 cites W1997643629 @default.
• W2064118841 cites W1999314849 @default.
• W2064118841 cites W2001525211 @default.
• W2064118841 cites W2014576184 @default.
• W2064118841 cites W2015176049 @default.
• W2064118841 cites W2016441306 @default.
• W2064118841 cites W2019742616 @default.
• W2064118841 cites W2022754746 @default.
• W2064118841 cites W2023038974 @default.
• W2064118841 cites W2026991751 @default.
• W2064118841 cites W2030185192 @default.
• W2064118841 cites W2035516048 @default.
• W2064118841 cites W2039493283 @default.
• W2064118841 cites W2040711852 @default.
• W2064118841 cites W2041756859 @default.
• W2064118841 cites W2044835337 @default.
• W2064118841 cites W2047063931 @default.
• W2064118841 cites W2052280808 @default.
• W2064118841 cites W2052645242 @default.
• W2064118841 cites W2055547342 @default.
• W2064118841 cites W2056697180 @default.
• W2064118841 cites W2059441263 @default.
• W2064118841 cites W2061133720 @default.
• W2064118841 cites W2067889691 @default.
• W2064118841 cites W2074292010 @default.
• W2064118841 cites W2076733230 @default.
• W2064118841 cites W2079255198 @default.
• W2064118841 cites W2086789117 @default.
• W2064118841 cites W2086840187 @default.
• W2064118841 cites W2086868295 @default.
• W2064118841 cites W2090959188 @default.
• W2064118841 cites W2093626313 @default.
• W2064118841 cites W2097365951 @default.
• W2064118841 cites W2097948220 @default.
• W2064118841 cites W2100837269 @default.
• W2064118841 cites W2102131116 @default.
• W2064118841 cites W2103774849 @default.
• W2064118841 cites W2103908315 @default.
• W2064118841 cites W2111297610 @default.
• W2064118841 cites W2128105883 @default.
• W2064118841 cites W2133524076 @default.
• W2064118841 cites W2136906322 @default.
• W2064118841 cites W2137588110 @default.
• W2064118841 cites W2140686653 @default.
• W2064118841 cites W2140704050 @default.
• W2064118841 cites W2149963584 @default.
• W2064118841 cites W2169217477 @default.
• W2064118841 cites W2169821755 @default.
• W2064118841 cites W235419956 @default.
• W2064118841 cites W50079648 @default.
• W2064118841 cites W2042597809 @default.
• W2064118841 doi "https://doi.org/10.1074/jbc.m509851200" @default.
• W2064118841 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16407225" @default.
• W2064118841 hasPublicationYear "2006" @default.
• W2064118841 type Work @default.
• W2064118841 sameAs 2064118841 @default.
• W2064118841 citedByCount "32" @default.
• W2064118841 countsByYear W20641188412012 @default.
• W2064118841 countsByYear W20641188412013 @default.
• W2064118841 countsByYear W20641188412014 @default.
• W2064118841 countsByYear W20641188412015 @default.

• next