The structural and photochemical changes in rhodopsin due to absorption of light are crucial for understanding the process of visual signaling. We investigated the structure of trans-retinal in the metarhodopsin I photointermediate (MI), where the retinylidene cofactor functions as an antagonist. Rhodopsin was regenerated using retinal that was (2)H-labeled at the C5, C9, or C13 methyl groups and was reconstituted with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Membranes were aligned by isopotential centrifugation, and rhodopsin in the supported bilayers was then bleached and cryotrapped in the MI state. Solid-state (2)H NMR spectra of oriented rhodopsin in the low-temperature lipid gel state were analyzed in terms of a static uniaxial distribution (Nevzorov, A. A.; Moltke, S.; Heyn, M. P.; Brown, M. F. J. Am. Chem. Soc. 1999, 121, 7636-7643). The line shape analysis allowed us to obtain the methyl bond orientations relative to the membrane normal in the presence of substantial alignment disorder (mosaic spread). Relative orientations of the methyl groups were used to calculate effective torsional angles between the three different planes that represent the polyene chain and the beta-ionone ring of retinal. Assuming a three-plane model, a less distorted structure was found for retinal in MI compared to the dark state. Our results are pertinent to how photonic energy is channeled within the protein to allow the strained retinal conformation to relax, thereby forming the activated state of the receptor.
Abstract:
The quadrupolar spin-lattice (T1) relaxation of deuterium labeled phospholipid bilayers has been investigated at a resonance frequency of 54.4 MHz. T1 measurements are reported for multilamellar dispersions, single bilayer vesicles, and chloroform/methanol solutions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), selectively deuterated at ten different positions in each of the fatty acyl chains and at the sn-3 carbon of the glycerol backbone. At all segment positions investigated, the T 1 relaxation times of the multilamellar and vesicle samples of DPPC were found to be similar. The profiles of the spin-lattice relaxation rate (1/T1) as a function of the deuterated chain segment position resemble the previously determined order profiles [A. Seelig and J. Seelig, Biochem. 13, 4839 (1974)]. In particular, the relaxation rates are approximately constant over the first part of the fatty acyl chains (carbon segments C3-C9), then decreasing in the central region of the bilayer. In chloroform/methanol solution, by contrast, the relaxation rates decrease continuously from the glycerol backbone region to the chain terminal methyl groups. The contributions from molecular order and motion to the T1 relaxation rates have been evaluated and correlation time profiles derived as a function of chain position. The results suggest that the motions of the various methylene segments are correlated in the first part of the fatty acyl chains (C3-C9), occurring at frequencies up to 1/τc∼1010Hz. Beyond C9, the rate and amplitude of the chain segmental motions increase, approaching that of simple paraffinic liquids in the central region of the bilayer (1/τc≃1011Hz). The T1 relaxation rates of multilamellar dispersions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) deuterated at the 9, 10 double bond of the sn-2 chain were also determined and found to be significantly faster than those of the CD2 chain segments of DPPC bilayers. This is most likely due to the larger size and correspondingly slower motion of the chain segment containing the double bond. At segments close to the lipid-water interface the rate of motion is considerably less than in the hydrocarbon region of the bilayer. © 1979 American Institute of Physics.
