Solid-State 2H NMR Investigation of Transducin Activation by Rhodopsin

Abstract

X-ray structures of active-state rhodopsin (Meta-II) and the cognate G-protein transducin are available, yet the transducin activation mechanism by rhodopsin is still obscure due to lack of atomistic dynamical information. We are studying the conformations of retinal in active Meta-II, and how the presence of the all-trans retinal agonist yields substantial differences in activation of transducin compared to opsin. Solid-state NMR spectroscopy gives information pertaining both to structures and dynamics, and is a powerful method to study how rhodopsin activates transducin. Experiments are currently underway with selectively deuterated retinal in aligned membrane samples containing active Meta-II [1]. Simulation of the 2H NMR lineshape of the aligned samples in terms of a static uniaxial distribution reveals the bond orientations of retinal methyl groups and mosaic spread, which represents the alignment disorder of the stacked membranes [2,3]. Comparison with the solid-state 2H NMR spectra predicated by published X-ray results enables proposed structures for active Meta-II to be tested [4]. Moreover, the solid-state 2H NMR spectral lineshapes show the role of dynamical fluctuations of the protein. We are also conducting solid-state 2H NMR experiments with deuterated C-terminal peptide of transducin to study the interaction between the G-protein and the rhodopsin transmembrane helices. Our hypothesis is that association and dissociation cycles of transducin depend on the relationship between the local dynamics of peptide and the fluctuations of rhodopsin helices. Solid state 2H NMR experiments can not only tell us how rhodopsin activates transducin, but also can reveal the general mechanisms whereby GPCRs activate the cognate G-proteins. [1] A.V. Struts et al. (2011) PNAS 108, 8263. [2] X. Xu et al. (2014) Encycl. Mag. Res. 3, 275-286. [3] B. Mertz et al. (2012) BBA 1818, 241-251. [4] A.V. Struts et al. (2007) JMB 372, 50-66.