Removal of the 9-methyl group of retinal inhibits signal transduction in the visual process. A fourier transform infrared and biochemical investigation

Abstract

The photoreaction of opsin regenerated with 9-demethylretinal has been investigated by UV-vis spectroscopy, flash photolysis experiments, and Fourier transform infrared difference spectroscopy. In addition, the capability of the illuminated pigment to activate the retinal G-protein has been tested. The photoproduct, which can be stabilized at 77 K, resembles more the lumirhodopsin species, and only minor further changes occur upon warming the sample to 170 K (stabilizing lumirhodopsin). UV-vis spectroscopy reveals no further changes at 240 K (stabilizing metarhodopsin I), but infrared difference spectroscopy shows that the protein as well as the chromophore undergoes further molecular changes which are, however, different from those observed for unmodified metarhodopsin I. UV-vis spectroscopy, flash photolysis experiments, and infrared difference spectroscopy demonstrate that an intermediate different from metarhodopsin II is produced at room temperature, of which the Schiff base is still protonated. The illuminated pigment was able to activate G-protein, as assayed by monitoring the exchange of GDP for GTP gamma S in purified G-protein, only to a very limited extent (approximately 8% as compared to rhodopsin). The results are interpreted in terms of a specific steric interaction of the 9-methyl group of the retinal in rhodopsin with the protein, which is required to initiate the molecular changes necessary for G-protein activation. The residual activation suggests a conformer of the photolyzed pigment which mimics metarhodopsin II to a very limited extent.

Chromophore structural changes in rhodopsin from nanoseconds to microseconds following pigment photolysis

“…The displacement of the chromophore was reduced by introducing twists along the single bonds C 14 OC 15 (40°), C 10 OC 11 (20°), and C 8 OC 9 (20°); whereas, the C 12 OC 13 twist was kept constant at 30°and the C 6 OC 7 angle was reduced to 20°to load the 5-methyl group against 8-H (3, 37). These twist assignments agree well with Raman and FTIR data (5,32). CCC bends of the chromophore in this intermediate (accompanied by CCCC torsions), leading to protein-retinal steric interactions, are thought to represent the main contribution to energy storage in the batho intermediate (7,43,44).…”

“…In batho, the newly introduced twist in the chromophore complicates calculation of the counterion-PSB distance. However, in bsi and lumi the chromophore twists have largely relaxed (31,32) and we assume that the relative shifts (numbers in parentheses in Table 1) of the max values, in proceeding from the dark state to bsi and from bsi to lumi, can be interpreted qualitatively in terms of counterion Ϫ PSB Intermediate spectra require the kinetic scheme to be known, and the bleach (the amount and Gaussian shape of the photolyzed sample determined in a separate experiment, data not shown) must be added back to the difference spectra (23). distance (24).…”

“…The Batho Intermediate. In batho, the SB environment remains unchanged (5,32), but the transition dipole orientation differs from the dark state by 8 Ϯ 2° (22). Taking the steric hindrance between the 13-methyl group and 10-H into account, isomerization must involve a rotation with the 13-methyl group moving away from the adjacent 10-H. To accomplish this, we moved the C 12 OH and C 11 OH bonds in opposite directions as previously proposed (7).…”

“…3 a and b shows the different conformations of the retinal in going from the dark state (green) to batho (red) as deduced from the above information and transition dipole orientations (22). The change of position and orientation of the SB proton after isomerization was minimized as required from FTIR and resonance Raman measurements (5,32). As a result, the largest chromophore movement is represented by the ring portion of the retinal.…”

A model for transmembrane helix with a cis‐Proline in the middle

“…Fax: (91) (80) 3341683. E-mail: sv@mbu.iisc.ernet.in **E-mail: laxman@mbu.iisc.ernet .in dopsin (bR) and Rhodopsin (Rh) associated with their function indicate the involvement of the peptide bond at the Pro residues as 'hinges' [27][28][29]. The findings of Strader et al, on Pro to Ser mutation in fl-adrenergic receptor had suggested the involvement of P323 in a receptor processing event such as folding [30].…”