Dynamics of Retinal Studied by 2H NMR Relaxation Underlie Multiscale Conformational Changes in Rhodopsin Activation

Abstract

The activation mechanism of the G protein-coupled receptor rhodopsin has been investigated through solid-state 2H NMR relaxation studies. Rhodopsin was regenerated with retinal 2H-labeled at the C5-, C9-, and C13-methyl groups and was recombined into POPC (dark state and Meta I) or POPC:DOPE (Meta II) [1-4]. The 2H NMR relaxation rates of Zeeman order (R1Z) or quadrupolar order (R1Q) were measured from −30 to −150°C in the dark, Meta I, and Meta II states. Relaxation data were interpreted in terms of axial rotation and off-axial motion of the methyl groups and revealed interactions between the retinal cofactor and the rhodopsin binding pocket. Relaxation rates show considerable mobility of the retinal ligand in the dark state despite the complete absence of basal activity. 2H NMR data indicate that the retinal C9-methyl group does not change its orientation upon photoisomerization. Rather it acts as a hinge for light-induced rotation of the C13-methyl toward the second extracellular loop E2. The C13-methyl rotation leads to a displacement of the E2 loop that is observed already in the Meta I state. Short T1Z relaxation times for the C5-methyl indicate that the β-ionone ring adopts a twisted 6-s-cis conformation, and remains in its hydrophobic binding pocket up to the Meta II state. An activation mechanism is proposed whereby the photonic energy is channeled by 11-cis to trans isomerization of retinal against the E2 loop by the C9- and C13-methyl groups, and toward helices H5 and H3 by the β-ionone ring and C5-methyl group.[1] G.F.J. Salgado et al. (2004) Biochemistry43, 12819. [2] G.F.J. Salgado et al. (2006) JACS128, 11067. [3] A.V. Struts et al. (2007) JMB372, 50. [4] M.F. Brown et al. (2009) Photochem. Photobiol.85, 442.