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Femtochemistry; fluorescence; photolysis; proteins; time-resolved spectroscopy
Abstract:
The photocycle of channelrhodopsin-2 is investigated in a comprehensive study by ultrafast absorption and fluorescence spectroscopy as well as flash photolysis in the visible spectral range. The ultrafast techniques reveal an excited-state decay mechanism analogous to that of the archaeal bacteriorhodopsin and sensory rhodopsin II from Natronomonas pharaonis. After a fast vibrational relaxation of the excited-state population with 150 fs its decay with mainly 400 fs is observed. Hereby, both the initial all-trans retinal ground state and the 13-cis-retinal K photoproduct are populated. The reaction proceeds with a 2.7 ps component assigned to cooling processes. Small spectral shifts are observed on a 200 ps timescale. They are attributed to conformational rearrangements in the retinal binding pocket. The subsequent dynamics progresses with the formation of an M-like intermediate (7 and 120 μs), which decays into red-shifted states within 3 ms. Ground-state recovery including channel closing and reisomerization of the retinal chromophore occurs in a triexponential manner (6 ms, 33 ms, 3.4 s). To learn more about the energy barriers between the different photocycle intermediates, temperature-dependent flash photolysis measurements are performed between 10 and 30°C. The first five time constants decrease with increasing temperature. The calculated thermodynamic parameters indicate that the closing mechanism is controlled by large negative entropy changes. The last time constant is temperature independent, which demonstrates that the photocycle is most likely completed by a series of individual steps recovering the initial structure.
