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J. Biol. Chem., Vol. 280, Issue 45, 38071-38080, November 11, 2005

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Heterotrimeric G-protein -Subunit Adopts a "Preactivated" Conformation When Associated with -Subunits^*

Najmoutin G. Abdulaev, Tony Ngo, Cheng Zhang, Andy Dinh, Danielle M. Brabazon¶, Kevin D. Ridge1, and John P. Marino2

From the Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and National Institute of Standards and Technology, Rockville, Maryland 20850, the Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, Texas 77030, and the ^¶Department of Chemistry, Loyola College in Maryland, Baltimore, Maryland 21210

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 -subunit (G) chimera (ChiT) associated with G-protein -subunit (G) and activated receptor (R^*) interactions. Here, we show that ChiT can be functionally reconstituted with G 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 G 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 (GTPS)/Mg²⁺-bound ¹⁵N-ChiT. As anticipated, G 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 - and GTPS/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 G ("preactivation") that may facilitate the interaction with R^* and subsequent GDP/GTP exchange.

Received for publication, May 12, 2005 , and in revised form, August 23, 2005.

^* This work was supported in part by Karl Kirchgessner Foundation and Robert A. Welch Foundation (AU-1613) awards (to K. D. R.) and Research Corporation Grant CC5250 (to D. M. B.). NMR instrumentation was supported in part by National Institutes of Health/National Center for Research Resources Grant RR015744, National Institute of Standards and Technology, and the W. M. Keck Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence may be addressed. Tel.: 713-500-5908; Fax: 713-500-0545; E-mail: Kevin.D.Ridge{at}uth.tmc.edu.

² To whom correspondence may be addressed. Tel.: 240-314-6160; Fax: 240-314-6255; E-mail: john.marino{at}nist.gov.

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