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Conformational Changes Associated With Receptor-Stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-Protein α-Subunit: NMR Analysis of the GTPγS-Bound States

Published

Author(s)

K D. Ridge, N G. Abdulaev, Xi-Cheng Zhang, T Ngo, D M. Brabazon, John P. Marino

Abstract

Solution NMR studies of a 15N-labeled G-protein ?-subunit (G?) chimera (15N-ChiT) reconstituted heterotrimer have previously shown that binding of ??-subunits induces a pre-activated conformation in G? that may facilitate interaction with R* and guanine nucleotide exchange (Abdulaev et al. (2005) J. Biol. Chem., in press). Here, we further demonstrate that the 15N-ChiT reconstituted heterotrimer can form functional complexes under NMR experimental conditions with light-activated, detergent solubilized rhodopsin, as well as a soluble mimic of R*. NMR methods are used to track light-activated rhodopsin triggered guanine nucleotide exchange and release of GTP?S/Mg2+-bound ChiT. An HSQC spectrum of activated GTP?S/Mg2+-bound ChiT reveals 1HN, 15N chemical shift changes relative to GDP/Mg2+-bound ChiT that are similar, but not identical, to those observed for the GDP AlF4-/Mg2+-bound state. Spectral line-widths observed for GTP?S/Mg2+-bound 15N-ChiT, however, indicate that it is more dynamic relative to the GDP/Mg2+- and GDP AlF4-/ Mg2+-bound states. In contrast to light-activated rhodopsin, a soluble mimic of R*, which does not catalytically interact with Gt (Abdulaev et al. (2000) J. Biol. Chem. 275, 39354-39363), is observed to form a stable complex with the GTP?S/Mg2+ exchanged heterotrimer. The HSQC spectrum of 15N-ChiT in this complex displays a unique 1HN, 15N chemical shift pattern that nonetheless shares similarities with both the GDP/Mg2+-bound heterotrimer and GTP?S/Mg2+-bound ChiT. Taken together, these findings demonstrate that R* induced changes in G? structure accompanying guanine nucleotide exchange can be followed by NMR and that guanine nucleotide exchange can be uncoupled from G??? dissociation.
Citation
Journal of Biological Chemistry
Volume
281
Issue
11

Citation

Ridge, K. , Abdulaev, N. , Zhang, X. , Ngo, T. , Brabazon, D. and Marino, J. (2006), Conformational Changes Associated With Receptor-Stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-Protein α-Subunit: NMR Analysis of the GTPγS-Bound States, Journal of Biological Chemistry (Accessed February 22, 2024)
Created March 17, 2006, Updated February 19, 2017