Conformational Changes at The Carboxyl Terminus of Gα Occur during G Protein Activation
1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês
10.1074/jbc.274.4.2379
ISSN1083-351X
AutoresChii‐Shen Yang, Nikolai P. Skiba, Maria Rosa Mazzoni, Heidi E. Hamm,
Tópico(s)Peptidase Inhibition and Analysis
ResumoTo understand the dynamics of conformational changes during G protein activation, surface exposed cysteine residues on Gα were fluorescently labeled. Limited trypsinolysis and mutational analysis of recombinant Gαt/Gαi1 determined that two cysteines are the major fluorescent labeling sites, Cys210, located in the switch II region, and Cys347 at the C terminus. Mutants with serines replacing Cys210 (Chi6a) and Cys347 (Chi6b) were single fluorescently labeled with lucifer yellow (LY), while a double mutant (Chi6ab) was no longer labeled. When Chi6b was labeled with LY on Cys210, AlF4− caused a 220% increase in LY fluorescence, indicating that the fluorescent group at Cys210 is a reporter of conformational change in the switch II region. Chi6a labeled at Cys347 also showed an AlF4−-dependent increase in LY fluorescence (91%), indicating that Gα activation leads to a conformational change at the COOH terminus. Preactivation of the protein with AlF4− before labeling led to a decreased incorporation of LY into Cys347suggesting that Gα activation buries Cys347. This COOH-terminal conformational change may provide the structural basis for communication between the GDP-binding site on Gα and activated receptors, and may contribute to dissociation of activated Gα subunit from activated receptor. To understand the dynamics of conformational changes during G protein activation, surface exposed cysteine residues on Gα were fluorescently labeled. Limited trypsinolysis and mutational analysis of recombinant Gαt/Gαi1 determined that two cysteines are the major fluorescent labeling sites, Cys210, located in the switch II region, and Cys347 at the C terminus. Mutants with serines replacing Cys210 (Chi6a) and Cys347 (Chi6b) were single fluorescently labeled with lucifer yellow (LY), while a double mutant (Chi6ab) was no longer labeled. When Chi6b was labeled with LY on Cys210, AlF4− caused a 220% increase in LY fluorescence, indicating that the fluorescent group at Cys210 is a reporter of conformational change in the switch II region. Chi6a labeled at Cys347 also showed an AlF4−-dependent increase in LY fluorescence (91%), indicating that Gα activation leads to a conformational change at the COOH terminus. Preactivation of the protein with AlF4− before labeling led to a decreased incorporation of LY into Cys347suggesting that Gα activation buries Cys347. This COOH-terminal conformational change may provide the structural basis for communication between the GDP-binding site on Gα and activated receptors, and may contribute to dissociation of activated Gα subunit from activated receptor. 125I-N-(3-indo-4-azidophenylpropionamido-S-(2-thiopyridyl)cysteine guanosine 5′-3-O-(thio)triphosphate lucifer yellow vinyl sulfone high performance liquid chromatography tosyl-l-lysine chloromethyl ketone. Heterotrimeric G proteins are activated by seven-transmembrane-spanning receptors and relay signals to downstream effectors, including cellular enzymes and ion channels. Upon agonist binding, receptors become activated and in turn interact with G proteins and catalyze GDP release from G protein α subunits. After the release of GDP, the Gα subunit, together with Gβγ subunits, remains in a tight complex with the receptor, which dissociates when GTP binds to the empty Gα subunit. Both the GTP-bound Gα subunit and the Gβγ subunit complex are then capable of regulating a variety of effectors on the intracellular face of the plasma membrane. The binding of Gα and Gβγ subunits is restored when the intrinsic GTPase activity in the Gα subunit hydrolyzes the bound GTP to GDP.In order to have a reliable method for studying the conformational changes in G proteins, we developed a fluorescent monitor because fluorescence is easily detectable and responsive to local environmental change. Cysteines are highly reactive and can be labeled with a variety of Cys-directed fluorescent groups. We determined the cysteines that are accessible to sulfhydryl-specific fluorescence labeling on Gα subunits and characterized the fluorescence changes on Gα when different sites are labeled. We replaced those accessible cysteines with serine to make a functionally cysteineless mutant in which to place additional cysteines at sites where we would like to monitor conformational changes or interaction with other proteins.To study structural changes in Gαt upon its activation, we used the functional derivative of Gαt, Gαt/Gαi1 chimera (Chi6) in which residues 216–294 of Gαt were replaced with the corresponding residues 220–298 from Gαi1. Chi6 can be conveniently expressed in Escherichia coli, and it was shown to have a similar rate of rhodopsin-catalyzed GDP/GTP exchange as Gαt does, implying that its receptor and Gβγ binding properties are Gαt-like (1Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The crystal structure of Chi6 in complex with Gβ1γ1 has been solved and revealed an identical geometry with wild type, native Gαt (2Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1044) Google Scholar). High expression levels of this protein inE. coli provided us milligram amounts of pure protein for biochemical and fluorescent studies as well as ease in constructing Gα mutants.Lucifer yellow, an environmentally sensitive fluorescent probe, was selected because it is a good reporter of local changes. We previously used this probe as a reporter of the binding of the inhibitory subunit of cGMP phosphodiesterase to Gαt(3Artemyev N.O. Rarick H.M. Mills J.S. Skiba N.P. Hamm H.E. J. Biol. Chem. 1992; 267: 25067-25072Abstract Full Text PDF PubMed Google Scholar).Ho and Fung (4Ho Y.-K. Fung B.K.-K. J. Biol. Chem. 1984; 259: 6694-6699Abstract Full Text PDF PubMed Google Scholar) previously reported that by using 5,5′-dithiobis-(2-nitrobenzoic acid) titration andN-ethylmaleimide modification, a total of five reactive sulfhydryls in native Gαt and nine reactive sulfhydryls in the SDS-denatured Gαt protein were found. Eight cysteines were found by cDNA sequencing of αt (5Yatsunami K. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4316-4320Crossref PubMed Scopus (85) Google Scholar). In 1988, by using125I-N-(3-indo-4-azidophenylpropionamido-S-(2-thiopyridyl)cysteine (125I-ACTP),1 a cross-linking reagent, Dhanasekaran et al. (6Dhanasekaran N. Wessling-Resnick M. Kelleher D.J. Johnson G.L. Ruoho A.E. J. Biol. Chem. 1988; 263: 17942-17950Abstract Full Text PDF PubMed Google Scholar) showed that Cys210 and Cys347 were the major reactive cysteines. Here, we show that the major labeling sites for LY are located on Cys210 and Cys347 in Gαt/Gαi chimeras. Also, we show that single labeling at either Cys210 or Cys347 can be used to report the local conformational changes around the labeled sites. As expected, Cys210 in the switch II region reports an AlF4−-dependent activating conformational change. Unexpectedly, there is also an AlF4−-dependent conformational change at Cys347, which may be important for allosteric communication between receptor binding and GDP-binding sites on the molecule.DISCUSSIONKnowledge of the functional structure of heterotrimeric G proteins has been greatly advanced with the solution of the crystal structure of all their subunits (2Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1044) Google Scholar, 11Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (521) Google Scholar, 12Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (702) Google Scholar, 13Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (529) Google Scholar, 14Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 265: 1405-1412Crossref PubMed Scopus (746) Google Scholar, 15Wall 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 (1004) Google Scholar, 16Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Crossref PubMed Scopus (707) Google Scholar, 17Sunahara R.K. Tesmer J.J. Gilman A.G. Sprang S.R. Science. 1997; 278: 1943-1947Crossref PubMed Scopus (262) Google Scholar, 18Tesmer J.J. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (667) Google Scholar). A comparison of the three-dimensional structure of the α subunits in their GDP, GTPγS, and transition state analogue forms has revealed the molecular principles of nucleotide binding, hydrolysis, and the nature of the conformation changes upon protein activation. The COOH-terminal region of Gαt, known to be important for receptor interaction (19West Jr., R.E. Moss J. Vaughan M. Lui T. Lin T.-Y. J. Biol. Chem. 1985; 260: 14428-14430Abstract Full Text PDF PubMed Google Scholar, 20Sullivan K.A. Miller R.T. Masters S.B. Beiderman B. Heideman W. Bourne H.R. Nature. 1987; 330: 758-760Crossref PubMed Scopus (161) Google Scholar, 21Hamm H.E. Deretic D. Arendt A. Hargrave P.A. Koenig B. Hofmann K.P. Science. 1988; 241: 832-835Crossref PubMed Scopus (390) Google Scholar, 22Simonds W.F. Goldsmith P.K. Woodard C.J. Unson C.G. Spiegel A.M. FEBS Lett. 1989; 249: 189-194Crossref PubMed Scopus (147) Google Scholar, 23Gutowski S. Smrcka A. Nowak L. Wu D. Simon M. Sternweis P.C. J. Biol. Chem. 1991; 266: 20519-20524Abstract Full Text PDF PubMed Google Scholar, 24Onrust R. Herzmark P. Chi P. Garcia P.D. Lichtarge O. Kingsley C. Bourne H.R. Science. 1997; 275: 381-384Crossref PubMed Scopus (196) Google Scholar), was not ordered in crystal structures (2Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1044) Google Scholar, 11Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (521) Google Scholar, 12Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (702) Google Scholar, 13Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (529) Google Scholar). Thus, other biochemical or biophysical methods are needed to further define the structural features of this region and understand its role in mediating receptor interaction and receptor induction of GDP release.In this report, we have developed an approach of sensitive real-time monitoring of the structural changes in Gα using targeted fluorescent labeling of single surface-exposed cysteine residues. Our studies have identified the labeling sites of LY on Gαt/Gαi1 chimera (Chi6), characterized each labeled protein and, most importantly, shown the existence of a previously unsuspected conformational change in the COOH terminus of Gα upon activation.The existence of two negative charges in LY and the mild conditions used for labeling Chi6 with LY allowed us to resolve differently modified proteins by anion-exchange chromatography (Fig. 2). The labeling sites on Gα identified in this report, Cys210and Cys347, confirms the studies of Dhanasekaran et al. (6Dhanasekaran N. Wessling-Resnick M. Kelleher D.J. Johnson G.L. Ruoho A.E. J. Biol. Chem. 1988; 263: 17942-17950Abstract Full Text PDF PubMed Google Scholar) who found that Cys347 is highly accessible and Cys210 is partially accessible to 125I-ACTP, when this cross-linking reagent was used to label Gαt.All mutants were properly folded and functional as judged by several criteria: first, all mutants had an increased Trp fluorescence upon addition of AlF4− indicating that they all have GDP bound and undergo conformational changes. Second, all mutants activated with AlF4− had similar affinity to Pγ when compared with Chi6 (1Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), which was in the range of 1 μm. Third, the cleavage patterns of tryptic digestion were identical in Chi6 and Chi6a and therefore it can be concluded the mutant chimeras were properly folded during expression.Our data clearly show that LY at Cys210 is a reporter of conformational change of the switch II region. The fluorescence of LY attached to Cys210 (Chi6a-LY) increases significantly (220%) in the presence of AlF4−. However, the intrinsic Trp fluorescence of Cys210-LY (Chi6b-LY) under similar conditions underwent only 1.2% change, which is much less than 50% change in Trp fluorescence for the unlabeled mutant. But Chi6b-LY protein does indeed undergo switch II region conformational change upon binding of AlF4− as judged by acquisition of high affinity for Pγ. An explanation for a decreased Trp fluorescence change in Cys210-LY may be that the fluorescent group at Cys210 increases local hydrophobicity in the environment of Trp207 that causes an increase in Trp fluorescence in the GDP-bound state of Gα. Upon addition of AlF4−, switch II moves and adopts the conformation of active Gα subunit, as Trp207 does. However, the increased hydrophobicity of Trp207 in the new environment is not much more than that created by the LY group, so that it does not result in a regular change of intrinsic fluorescence.The solvent-accessible surface area of Cys210 decreased 68.6% in the activated form of Gαt. This calculation matches well with our finding that the labeling efficiency at Cys210 dropped 77.8% in the presence of AlF4−. Thus, Cys210 of Chi6b becomes partially protected from LY labeling in the presence of AlF4−, reflecting these changes in accessibility. The method is thus clearly useful for monitoring known conformational changes.The conformational changes seen at the COOH terminus of Gα are the first indications that the GTP-dependent or activation-dependent conformational switch may extend to this region of the molecule. Several facts support the existence of those environmental changes around the COOH terminus. First, the fluorescence of LY at Cys347-LY (Chi6a-LY) increases by more than 91% in the presence of AlF4− and can be reversed by 10 mm EDTA. There is no nonspecific interaction between LY and AlF4− since in the presence of free LY, no fluorescence change was observed by addition of sodium fluoride and AlCl3 (data not shown). Second, the efficiency of Cys347 labeling with LY for Chi6a is significantly reduced with addition of AlF4−, suggesting that surface exposure and reactivity of this residue is decreased as a result of protein activation. To test whether a fluorescent tag on a cysteine in a part of the molecule that is known to have no conformation change upon activation, Chi6ab, with both reactive cysteines changed to serines, was mutated to replace Val301 with cysteine. There was no AlF4−-dependent fluorescent change in the resultant mutant, Chi6ab-Cys301-LY (data not shown). Thus the fluorescence change is not seen in regions known not to change conformation. Third, these effects are very similar to those we observed for Cys210 in Chi6b, which is an indicator of the known conformational change of switch II region. Thus, we can conclude that it is likely that a change in conformation does exist at or near the COOH terminus of Gα upon activation or more precisely in the GDP-AlF4− form. The comparison of the kinetics of fluorescence changes monitored by LY to the fluorescent changes monitored by Trp207 for Chi6a-LY upon addition of AlF4− shows that they have similar time course (Fig. 4 C). This analysis implies that the structural change at the COOH terminus is rate limited by the relatively slow movement of the switch II region upon binding of AlF4−. Dhanasekaran et al.(1988) also proposed that conformational changes could be transmitted between domains containing Cys347 and Cys210 in Gαt because the photoactivation of the phenylazide moiety of 125I-ACTP labeled at Cys347 caused an insertion to the 12-kDa fragment (residues Arg204-Arg310), which contains Cys210. Our data suggest that LY-bound Cys347is in a more hydrophobic environment in GDP-AlF4− than in the GDP-bound Gα. Another observation is the Cys347 is the major labeling site for Chi6 and the labeling of Cys347 reduces the labeling efficiency of Cys210, which confirms the result suggested in 125I-ACTP labeling study (6Dhanasekaran N. Wessling-Resnick M. Kelleher D.J. Johnson G.L. Ruoho A.E. J. Biol. Chem. 1988; 263: 17942-17950Abstract Full Text PDF PubMed Google Scholar). This leads us to speculate that the COOH terminus of Gα does not interact with the α2/β4 loop in Gα-GDP form, but in the active conformation the α2/β4 loop and COOH terminus of Gα move closer.What is the nature of this COOH-terminal conformational change? According to the crystal structure of Gα-GTPγS (12Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (702) Google Scholar), in two out of three molecules in the asymmetric unit, the final eight residues are disordered and not visible while in a third molecule, the COOH-terminal residues 343–349 of Gαt can be seen. The residues 343–349 make van der Waals contacts with residues 212–215 of the α2/β4 loop. It is not clear whether this is a crystal packing artifact or an indication of one possible orientation of the COOH terminus.In Gαt, Asn343 is the last residue that is consistently seen. It is ∼10 Å from the α2/β4 loop which is part of Switch II. Two possibilities exist: 1) the conformation of the COOH-terminal region changes upon activation, 2) the COOH terminus itself does not undergo a conformational change, but its environment changes. For example, the disordered COOH-terminal region including Cys347 is close to the α2/β4 loop and in the active conformation a binding pocket for the COOH terminus opens up near the switch II region such that Cys347 becomes more buried. In either case, the protection from fluorescent labeling by preactivation, and the activation-dependent fluorescent change indicates an important communication between the GDP binding pocket and the COOH terminus of the protein.The COOH-terminal conformational change that was detected in this study is of significant interest because of the implications for receptor interaction mechanisms. The COOH terminus of Gα is known to be a key determinant of the fidelity of receptor activation (19West Jr., R.E. Moss J. Vaughan M. Lui T. Lin T.-Y. J. Biol. Chem. 1985; 260: 14428-14430Abstract Full Text PDF PubMed Google Scholar, 23Gutowski S. Smrcka A. Nowak L. Wu D. Simon M. Sternweis P.C. J. Biol. Chem. 1991; 266: 20519-20524Abstract Full Text PDF PubMed Google Scholar). Since known receptor-binding regions on Gα are distant from the GDP-binding site, it is likely that an allosteric mechanism triggers GDP release (24Onrust R. Herzmark P. Chi P. Garcia P.D. Lichtarge O. Kingsley C. Bourne H.R. Science. 1997; 275: 381-384Crossref PubMed Scopus (196) Google Scholar). It appears from a number of studies that mutations at the COOH terminus of Gα proteins can both regulate specific receptor interaction and affect GDP affinity (24Onrust R. Herzmark P. Chi P. Garcia P.D. Lichtarge O. Kingsley C. Bourne H.R. Science. 1997; 275: 381-384Crossref PubMed Scopus (196) Google Scholar, 25Denker B.M. Schmidt C.J. Neer E.J. J. Biol. Chem. 1992; 267: 9998-10002Abstract Full Text PDF PubMed Google Scholar, 26Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (601) Google Scholar, 27Osawa S. Weiss E.R. J. Biol. Chem. 1995; 270: 31052-31058Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 28Conklin B.R. Herzmark P. Ishida S. Voyno-Yasenetskaya T.A. Sun Y. Farfel Z. Bourne H.R. Mol. Pharmacol. 1996; 50: 885-890PubMed Google Scholar). COOH-terminal peptides from Gα can both block receptor-G protein interaction and stabilize the active conformation of G protein-coupled receptors (21Hamm H.E. Deretic D. Arendt A. Hargrave P.A. Koenig B. Hofmann K.P. Science. 1988; 241: 832-835Crossref PubMed Scopus (390) Google Scholar, 29Dratz E.D. Fursteneau J.E. Lambert C.G. Thireault D.L. Rarick H. Schepers T. Pakhlevaniants S. Hamm H.E. Nature. 1993; 363: 276-280Crossref PubMed Scopus (151) Google Scholar, 30Rasenick M.M. Watanabe M. Lazarevic M.B. Hatta S. Hamm H.E. J. Biol. Chem. 1994; 269: 21519-21525Abstract Full Text PDF PubMed Google Scholar, 31Martin E.L. Rens-Domiano S. Schatz P.J. Hamm H.E. J. Biol. Chem. 1996; 271: 361-366Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 32Gilchrist A. Mazzoni M.R. Dineen B. Dice A. Linden J. Proctor W.R. Lupica C.R. Dunwiddie T.V. Hamm H.E. J. Biol. Chem. 1998; 273: 14912-14919Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). These data suggest that the COOH terminus may be a key relay for communication between the activated receptor and the GDP binding pocket. The activation-dependent conformational change reported here thus suggests specific regulation of receptor affinity by the status of the guanine nucleotide bound in the binding pocket. It is known that when receptor, ligand, and G protein are bound, GDP release leads to a high affinity "ternary complex" which only slowly dissociates without guanine nucleotide binding (33Iyengar R. Abramowitz J. Bordelon-Riser M. Blume A.J. Birnbaumer L. J. Biol. Chem. 1980; 255: 10312-10321Abstract Full Text PDF PubMed Google Scholar, 34Pfister C. Kuhn H. Chabre M. Eur. J. Biochem. 1983; 136: 489-499Crossref PubMed Scopus (44) Google Scholar, 35Birnbaumer L. Codina J. Mattera R. Cerione R.A. Hildebrandt J.D. Sunyer T. Rojas F.J. Caron M.G. Lefkowitz R.J. Iyengar R. Recent Prog. Horm. Res. 1985; 41: 41-99PubMed Google Scholar, 36Kahlert M. Konig B. Hofmann K.P. J. Biol. Chem. 1990; 265: 18928-18932Abstract Full Text PDF PubMed Google Scholar, 37Panico J. Parkes J.H. Liebman P.A. J. Biol. Chem. 1990; 265: 18922-18927Abstract Full Text PDF PubMed Google Scholar). It is possible that an activation-dependent conformational change at the COOH terminus of Gα might lead to a lowered receptor affinity and dissociation from the ternary complex. Currently, it is thought that dissociation of activated G protein from receptor is secondary to the GTP-dependent dissociation of Gα from Gβγ. This concept has not been rigorously tested, however. Future studies will test the notion that the COOH-terminal conformational change after GTP binding is a component of G protein dissociation from an activated receptor.Site-specific Cys-directed fluorescent groups could be reporters for conformation changes in various regions of Gα. For example, they could probe conformational changes in other regions of the Gα subunit like the NH2 terminus, which was not resolved in crystal structures of either the free Gα GDP form (11Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (521) Google Scholar) or the GTPγS form (12Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (702) Google Scholar, 14Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 265: 1405-1412Crossref PubMed Scopus (746) Google Scholar). Also, they could be used to monitor protein-protein interaction, such as Gα interaction with receptors, Gβγ, and effectors. Future studies will explore these possibilities. Heterotrimeric G proteins are activated by seven-transmembrane-spanning receptors and relay signals to downstream effectors, including cellular enzymes and ion channels. Upon agonist binding, receptors become activated and in turn interact with G proteins and catalyze GDP release from G protein α subunits. After the release of GDP, the Gα subunit, together with Gβγ subunits, remains in a tight complex with the receptor, which dissociates when GTP binds to the empty Gα subunit. Both the GTP-bound Gα subunit and the Gβγ subunit complex are then capable of regulating a variety of effectors on the intracellular face of the plasma membrane. The binding of Gα and Gβγ subunits is restored when the intrinsic GTPase activity in the Gα subunit hydrolyzes the bound GTP to GDP. In order to have a reliable method for studying the conformational changes in G proteins, we developed a fluorescent monitor because fluorescence is easily detectable and responsive to local environmental change. Cysteines are highly reactive and can be labeled with a variety of Cys-directed fluorescent groups. We determined the cysteines that are accessible to sulfhydryl-specific fluorescence labeling on Gα subunits and characterized the fluorescence changes on Gα when different sites are labeled. We replaced those accessible cysteines with serine to make a functionally cysteineless mutant in which to place additional cysteines at sites where we would like to monitor conformational changes or interaction with other proteins. To study structural changes in Gαt upon its activation, we used the functional derivative of Gαt, Gαt/Gαi1 chimera (Chi6) in which residues 216–294 of Gαt were replaced with the corresponding residues 220–298 from Gαi1. Chi6 can be conveniently expressed in Escherichia coli, and it was shown to have a similar rate of rhodopsin-catalyzed GDP/GTP exchange as Gαt does, implying that its receptor and Gβγ binding properties are Gαt-like (1Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The crystal structure of Chi6 in complex with Gβ1γ1 has been solved and revealed an identical geometry with wild type, native Gαt (2Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1044) Google Scholar). High expression levels of this protein inE. coli provided us milligram amounts of pure protein for biochemical and fluorescent studies as well as ease in constructing Gα mutants. Lucifer yellow, an environmentally sensitive fluorescent probe, was selected because it is a good reporter of local changes. We previously used this probe as a reporter of the binding of the inhibitory subunit of cGMP phosphodiesterase to Gαt(3Artemyev N.O. Rarick H.M. Mills J.S. Skiba N.P. Hamm H.E. J. Biol. Chem. 1992; 267: 25067-25072Abstract Full Text PDF PubMed Google Scholar). Ho and Fung (4Ho Y.-K. Fung B.K.-K. J. Biol. Chem. 1984; 259: 6694-6699Abstract Full Text PDF PubMed Google Scholar) previously reported that by using 5,5′-dithiobis-(2-nitrobenzoic acid) titration andN-ethylmaleimide modification, a total of five reactive sulfhydryls in native Gαt and nine reactive sulfhydryls in the SDS-denatured Gαt protein were found. Eight cysteines were found by cDNA sequencing of αt (5Yatsunami K. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4316-4320Crossref PubMed Scopus (85) Google Scholar). In 1988, by using125I-N-(3-indo-4-azidophenylpropionamido-S-(2-thiopyridyl)cysteine (125I-ACTP),1 a cross-linking reagent, Dhanasekaran et al. (6Dhanasekaran N. Wessling-Resnick M. Kelleher D.J. Johnson G.L. Ruoho A.E. J. Biol. Chem. 1988; 263: 17942-17950Abstract Full Text PDF PubMed Google Scholar) showed that Cys210 and Cys347 were the major reactive cysteines. Here, we show that the major labeling sites for LY are located on Cys210 and Cys347 in Gαt/Gαi chimeras. Also, we show that single labeling at either Cys210 or Cys347 can be used to report the local conformational changes around the labeled sites. As expected, Cys210 in the switch II region reports an AlF4−-dependent activating conformational change. Unexpectedly, there is also an AlF4−-dependent conformational change at Cys347, which may be important for allosteric communication between receptor binding and GDP-binding sites on the molecule. DISCUSSIONKnowledge of the functional structure of heterotrimeric G proteins has been greatly advanced with the solution of the crystal structure of all their subunits (2Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1044) Google Scholar, 11Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (521) Google Scholar, 12Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (702) Google Scholar, 13Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (529) Google Scholar, 14Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 265: 1405-1412Crossref PubMed Scopus (746) Google Scholar, 15Wall 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 (1004) Google Scholar, 16Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Crossref PubMed Scopus (707) Google Scholar, 17Sunahara R.K. Tesmer J.J. Gilman A.G. Sprang S.R. Science. 1997; 278: 1943-1947Crossref PubMed Scopus (262) Google Scholar, 18Tesmer J.J. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (667) Google Scholar). A comparison of the three-dimensional structure of the α subunits in their GDP, GTPγS, and transition state analogue forms has revealed the molecular principles of nucleotide binding, hydrolysis, and the nature of the conformation changes upon protein activation. The COOH-terminal region of Gαt, known to be important for receptor interaction (19West Jr., R.E. Moss J. Vaughan M. Lui T. Lin T.-Y. J. Biol. Chem. 1985; 260: 14428-14430Abstract Full Text PDF PubMed Google Scholar, 20Sullivan K.A. Miller R.T. Masters S.B. Beiderman B. Heideman W. Bourne H.R. Nature. 1987; 330: 758-760Crossref PubMed Scopus (161) Google Scholar, 21Hamm H.E. Deretic D. Arendt A. Hargrave P.A. Koenig B. Hofmann K.P. Science. 1988; 241: 832-835Crossref PubMed Scopus (390) Google Scholar, 22Simonds W.F. Goldsmith P.K. Woodard C.J. Unson C.G. Spiegel A.M. FEBS Lett. 1989; 249: 189-194C
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