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|Author(s):||K D. Ridge; N G. Abdulaev; Xi-Cheng Zhang; T Ngo; D M. Brabazon; John P. Marino;|
|Title:||Conformational Changes Associated With Receptor-Stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-Protein α-Subunit: NMR Analysis of the GTPγS-Bound States|
|Published:||March 17, 2006|
|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|
|Pages:||pp. 7635 - 7648|
|Research Areas:||NMR, Chemical Analysis|