Preparation of metarhodopsin I / equipment used:
Rho( POPC) ( 1: 50 molar ratio) was prepared in MES buffer, pH range 6, 5-7 and oriented on ultra thin glass plates ( 6x12 mm), the sample was placed in a glove box at 20C under gentle stream of argon to protect lipids from oxidation. After dehydration metarhodopsin(I) was produced by illumination for 1-2 min. at 20C with green light.
Figure1 representing UV- visible spectra of oriented samples used for 2H NMR spectroscopy.
Aligned sample are showing Rhodopsin POPC (1:50)
represented with 11-Cis retinal labeled at a. C5, b. C9 and c. C13.
Figure1 shows the photolysed sample that gave a peak at λmax ≈ 479 which represents inactive MI, the peak shown at λmax = 500 is due to the presence of nonbleached Rhodopsin and residues this peak will not show after the refinement of the conditions. The final step before using the sample for 2H NMR is stacking the samples in a cutoff 8x 22 mm NMR tube and chilled to – 70 and sealed with Teflon plug. Solid state 2H NMR spectra used 76.77 MHz from Bruker AMX-500 spectrometer.
Rhodopsin upon illumination:
Illumination causes conformational changes of the highly strained Rhodopsin. The Retinal is consisting of β-ionone ring and the retinylindene chain. Figure2 shows 2H NMR spectra for aligned
a. <!--[endif]-->MI/POPC and
b.unbleached Rho/POPC (1:50) to study and determined the bond orientation for the retinylidene C5, C9, C13 methyl groups.
Figure2 Solid-state 2H NMR spectra for aligned (a) MI/POPC and (b) Rho/POPC (1:50) membranes in MES
buffer at pH 7. Data were acquired at – 100oC for MI and -150oC for the dark state at zero
tilt (θ = 0o) for C5, C9, and C13 2H-labled methyl groups. Experimental
spectra (black) are shown with theoretical simulations (red) and residuals (blue).
Three calculated 2HNMR line shapes are distinguished; experimental line (black), theoretical line (red), and the residual line (blue). The theoretical line shape depends on θB and the alignment disorder (mosaic spread) σ.
In comparing fig la to fig 1b (the dark state), C9 methyl is distinguished and the difference was explained by the fact that σ (alignment disorder) in the dark was (18-210) vs. σ = 22-250 for the MI sample i.e. The difference in shape was due to the increase in σ rather than θB (bond orientation). This fact indicates the importance of alignment disorder.
Figure3. Is another display of the spectra of C5, C9, C13 as function of θB and σ and also showing the root- mean-square-deviation (RMSD) error surface. The figure shows a nonsymmetrical surface error for C5 mettyl which indicates that β-ionone ring could have different conformation affecting the 2HNMR angles.
<!--[if !vml]--><!--[endif]--> Figure3 Error surfaces for MI state calculated from RMSD analysis of simulated vs experimental 2H NMR spectra for (a) C5, (b) C9,
and (c) C13 methyl groups. Methyl bond orientation θB and mosaic spread (σ) are free parameters
and random from 0o to 90o. Floors of the three surfaces indicate the minima in the RMSD corresponding
to the best fit values of θB and mosaic spread (σ).
Table 1 show the most significant change in θB is with C13 When photoisomerization from 11-cis to all trance takes place.
Knowing the orientation and the conformation of retinal within the MI binding pocket is important for two reasons:
a. When isomerization of the retinal occurs, it will be possible to mathematically calculate energy stored in vision.