Photo-isomerization of the 11-cis retinal chromophore activates the mammalian light-receptor rhodopsin, a representative member of a major superfamily of transmembrane G-protein-coupled receptor proteins (GPCRs) responsible for many cell signal communication pathways. Although low-resolution (5 A) electron microscopy studies confirm a seven transmembrane helix bundle as a principal structural component of rhodopsin, the structure of the retinal within this helical bundle is not known in detail. Such information is essential for any theoretical or functional understanding of one of the fastest occurring photoactivation processes in nature, as well as the general mechanism behind GPCR activation. Here we determine the three-dimensional structure of 11-cis retinal bound to bovine rhodopsin in the ground state at atomic level using a new high-resolution solid-state NMR method. Significant structural changes are observed in the retinal following activation by light to the photo-activated M(I) state of rhodopsin giving the all-trans isomer of the chromophore. These changes are linked directly to the activation of the receptor, providing an insight into the activation mechanism of this class of receptors at a molecular level.
We present the first application of MAOSS (magic angle oriented sample spinning) NMR spectroscopy
to a large membrane protein. This new solid-state NMR approach is used to study the orientation of the deuterated
methyl group in [18-CD3]-retinal in oriented bacteriorhodopsin in both the photocycle ground state (bR568)
and in the photo intermediate state M412. Deuterium MAS spectra consist of a set of narrow spinning sidebands
if the sample spinning rate does not exceed the anisotropy of the quadrupole interaction. In ordered systems,
such as proteins in oriented membranes, each sideband intensity is orientationally dependent. The observed
MAS sideband pattern is modulated in a highly sensitive way by changes in the molecular orientation of the
CD3 group during the transition from all-trans- to 13-cis-retinal upon photoactivation. The significant
improvement in spectral sensitivity and resolution, compared to static NMR on oriented samples, allows a
reliable and precise data analysis even from lower spin concentrations and has more general consequences for
studying oriented membrane proteins by NMR. MAOSS NMR is shown to be a feasible method for the accurate
determination of local molecular orientations in large molecular systems which are currently a challenge for
crystallography.
Rhodopsin is the retinal photoreceptor responsible for visual signal transduction. To determine the orientation and conformation of retinal within the binding pocket of this membrane bound receptor, an ab initio solid state 2 H NMR approach was used. Bovine rhodopsin containing 11-cis retinal, specifically deuterated at its methyl groups at the C 19 or C 20 position, was uniaxially oriented in DMPC bilayers. Integrity of the membranes and quality of alignment were monitored by 31 P NMR. Analysis of the obtained 2 H NMR spectra provided angles for the individual labelled chemical bond vectors leading to an overall picture for the three dimensional structure of the polyene chain of the chromophore in the protein binding pocket around the Schiff base attachment site.z 1998 Federation of European Biochemical Societies.
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