Vibrational Spectrum of the Lumi Intermediate in the Room Temperature Rhodopsin Photo-Reaction

Abstract

The vibrational spectrum (650-1750 cm(-1)) of the lumi-rhodopsin (lumi) intermediate formed in the microsecond time regime of the room-temperature rhodopsin (RhRT) photoreaction is measured for the first time using picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). The vibrational spectrum of lumi is recorded 2.5 micros after the 3-ps, 500-nm excitation of RhRT. Complementary to Fourier transform infrared spectra recorded at Rh sample temperatures low enough to freeze lumi, these PTR/CARS results provide the first detailed view of the vibrational degrees of freedom of room-temperature lumi (lumiRT) through the identification of 21 bands. The exceptionally low intensity (compared to those observed in bathoRT) of the hydrogen out-of-plane (HOOP) bands, the moderate intensity and absolute positions of C-C stretching bands, and the presence of high-intensity C==C stretching bands suggest that lumiRT contains an almost planar (nontwisting), all-trans retinal geometry. Independently, the 944-cm(-1) position of the most intense HOOP band implies that a resonance coupling exists between the out-of-plane retinal vibrations and at least one group among the amino acids comprising the retinal binding pocket. The formation of lumiRT, monitored via PTR/CARS spectra recorded on the nanosecond time scale, can be associated with the decay of the blue-shifted intermediate (BSI(RT)) formed in equilibrium with the bathoRT intermediate. PTR/CARS spectra measured at a 210-ns delay contain distinct vibrational features attributable to BSI(RT), which suggest that the all-trans retinal in both BSI(RT) and lumiRT is strongly coupled to part of the retinal binding pocket. With regard to the energy storage/transduction mechanism in RhRT, these results support the hypothesis that during the formation of lumiRT, the majority of the photon energy absorbed by RhRT transfers to the apoprotein opsin.

Chromophore Structure in Lumirhodopsin and Metarhodopsin I by Time-Resolved Resonance Raman Microchip Spectroscopy

“…This ethylenic band is consistent with the 492 nm absorption band maximum for Lumi (4), based on the correlation of the ethylenic frequencies of retinals with their absorption maxima (27). An ethylenic mode at 1548 cm -1 was also observed in the Lumi CARS spectrum (22). In the fingerprint region, five bands at 1155, 1205, 1214 (sh), 1242, and 1289 cm -1 are observed.…”

Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system

“…Many techniques for the study of rhodopsin and other receptors [such as UV\visible, fluorescence, ESR and Fourier-transform infrared (FTIR) spectroscopies, G-protein activation and signal pathway assays] require small or only moderate amounts of material. However, crystallization attempts and powerful biophysical techniques, such as Raman spectroscopy and time-resolved UV\visible or FTIR spectroscopy to study ultrafast kinetics and structural changes, or solid-state NMR spectroscopy to determine structural constraints at high resolution, require large amounts of material (up to tens of milligrams) [7,8]. So far, the availability of only limited amounts of recombinant protein has been the major limitation in applying these techniques.…”

Coherent Anti-Stokes Raman Scattering (CARS) Microscopy: A Novel Technique for Imaging the Retina

“…Previous studies have also used CARS and TPAF to study the cycle of 11-cis retinol diffusion and lightinduced photoisomerization in isolated rod outer segments. 43,44 The work presented here may subsequently lead to in vivo imaging experiments which monitor the recycling of retinol within the living mouse eye, giving further insight into retinal function and dysfunction.…”