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(The FASEB Journal. 2007;21:782.3)
© 2007 FASEB
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782.3

Probing the molecular mechanism of heterotrimeric G-protein signaling using high-resolution NMR

Najmoutin G Abdulaev1, Eva Ramon2, Tony Ngo2, Danielle M Brabazon3, John P Marino1 and Kevin D Ridge2

1 Center for Advanced Research in Biotechnology, NIST and UMBI, 9600 Gudelsky Drive, Rockville, MD, 20850,
2 Center for Membrane Biology, UTHSC-Houston, 6431 Fannin Street, Houston, TX, 77030,
3 Department of Chemistry, Loyola College in Maryland, 4501 N. Charles Street, Baltimore, MD, 21210

ABSTRACT

The structural basis for activation of heterotrimeric ({alpha} ß {gamma}) G-proteins by G-protein coupled receptors (GPCRs) is not well understood. Using the rhodopsin/transducin system, conformational changes in G{alpha} accompanying heterotrimer formation and activated GPCR (R*) catalyzed GDP/GTP exchange are being tracked by high-resolution NMR. We have shown that

  1. an 15N-labeled G{alpha} chimera (ChiT) that displays relatively well-dispersed 2D spectra and uniform line widths undergoes aluminum fluoride induced perturbations in switch II and at the C-terminus;
  2. some anticipated R*- and GTP-induced perturbations in these same regions are already evident upon heterotrimer formation, likely providing kinetic advantages in R*/G-protein coupling;
  3. R*- and Gß {gamma}-released exchanged ChiT displays further C-terminal perturbation and increased conformational flexibility of switch II, which may be important for G{alpha}/effector interactions and GTP hydrolysis;
  4. the GDP-released, R*-bound state of ChiT shows severe line broadening suggestive of a dynamic intermediate that results from changes in the R*-interacting N- and C-termini; and
  5. N- and C-terminal mutations can have long-range consequences on ChiT conformation, particularly in switch II.

These results provide new insights into the mechanism of signal propagation from R* to the G-protein.

Support by NIH, Robert A. Welch Foundation, and Spanish Ministry of Science.





This Article
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