Time-Resolved Resonance Raman Analysis of Chromophore Structural Changes in the Formation and Decay of Rhodopsins BSI Intermediate

摘要

Time-resolved resonance Raman microchip flow experiments are performed to obtain the vibrational spectrum of the chromophore in rhodopsin's BSI intermediate and to probe structural changes in the bathorhodopsin-to-BSI and BSI-to-lumirhodopsin transitions. Kinetic Raman spectra from 250 ns to 3 μs identify the key vibrational features of BSI. BSI exhibits relatively intense HOOP modes at 886 and 945 cm^-1 that are assigned to C14H and C11H=C12H Au wags, respectively. This result suggests that in the bathorhodopsin-to-BSI transition the highly strained all-trans chromophore has relaxed in the C10-C11=C12-C13 region, but is still distorted near C14. The low frequency of the 11,12 Au HOOP mode in BSI compared with that of lumirhodopsin and metarhodopsin I idicates weaker coupling between the 11H and 12H wags due to residual distortion of the BSI chromphore near C11=C12. The C=NH⁺ stretching mode in BSI at 1653 cm^-1 exhibits a normal deuteriation indused downshift of 23 cm^-1 implying that there is no significant structural rearrangement of the Schiff base counterion region in the transition of bathorhodopsin to BSI. However, a dramatic Schiff base environment change occurs in the BSI-to-lumirhodipsin transition. because the 1638 cm^-1 C=NH⁺ stretching mode in lumirhodopsin is unussully low and shifts only 7cm^-1 in D2O, suggesting that it has essentially no H-bonding acceptor. With these data we can for the first time compare and discuss the room temperature resonance Raman vibrational structure of all the key intermediates in visual excitation.