Crystal structures of rhodopsin are available, yet details of the activation mechanism remain unknown [1,2]. We applied solid-state 2H NMR to investigate structural and dynamical changes occurring in the process of rhodopsin activation. From the 2H NMR spectra, molecular mobility can be obtained by calculating the segmental order parameters from the residual quadrupolar couplings (RQCs). Moreover 2H nuclear spin relaxation rates related to the dynamics can be measured together with the RQCs [2]. Site-specific 2H labels were introduced into different methyl groups of retinal, and relaxation rate measurements were performed as a function of temperature (−30 to −150°C) [3]. Model-free analysis employed an irreducible representation of the combined 2H NMR line shape and relaxation data. Fluctuations of the irreducible components with respect to the average values are characterized by the individual spectral densities of motion evaluated at characteristic frequencies: J0(0), J1(ω0), and J2(2ω0), where ω0 is the nuclear resonance frequency [4]. Differences in the spectral densities manifest details of the methyl group motions within the retinal binding pocket at low temperature. At the high temperature limit, J1(ω0) and J2(2ω0) are insensitive to details of motion and collapse to a universal curve, thus substantiating the validity of the model-free analysis. Further analysis of J1(ω0) and J2(2ω0) involved simultaneous temperature-dependent fitting in terms of both diffusion and jump models for the methyl group dynamics. We conclude that spectral density analysis in terms of fluctuations of nuclear spin Hamiltonian will help us understand the activation mechanism and molecular dynamics of rhodopsin and related G protein-coupled receptors.[1] A.V. Struts et al. (2011) PNAS 108, 8263-8268. [2] M.F. Brown et al. (2010) BBA 1798, 177-193. [3] A.V. Struts et al. (2011) NSMB 18, 392-394. [4] M.F. Brown (1982) JCP 77, 1576-1599.