Abstract
Channelrhodopsin-2 (ChR2) is a microbial type rhodopsin and a light-gated cation channel that controls phototaxis in Chlamydomonas. We expressed ChR2 in COS-cells, purified it, and subsequently investigated this unusual photoreceptor by flash photolysis and UV-visible and Fourier transform infrared difference spectroscopy. Several transient photoproducts of the wild type ChR2 were identified, and their kinetics and molecular properties were compared with those of the ChR2 mutant E90Q. Based on the spectroscopic data we developed a model of the photocycle comprising six distinguishable intermediates. This photocycle shows similarities to the photocycle of the ChR2-related Channelrhodopsin of Volvox but also displays significant differences. We show that molecular changes include retinal isomerization, changes in hydrogen bonding of carboxylic acids, and large alterations of the protein backbone structure. These alterations are stronger than those observed in the photocycle of other microbial rhodopsins like bacteriorhodopsin and are related to those occurring in animal rhodopsins. UV-visible and Fourier transform infrared difference spectroscopy revealed two late intermediates with different time constants of tau = 6 and 40 s that exist during the recovery of the dark state. The carboxylic side chain of Glu(90) is involved in the slow transition. The molecular changes during the ChR2 photocycle are discussed with respect to other members of the rhodopsin family.
Highlights
Channelrhodopsins (ChRs)3 are light-gated cation channels [1, 2] that share homology with other microbial rhodopsins such as bacteriorhodopsin (BR), halorhodopsin (HR), and sensory rhodopsin (SR)
A recent model for recombinant Volvox channelrhodopsin (VChR), purified from green monkey COS cells, comprises the two dark states D470 and D480, characterized by a fine structured UVvisible absorption spectrum with maxima at 470 and 480 nm, respectively [14]. These two states, which exist in a pH-dependent equilibrium, are both converted by light via retinal isomerization and transient Schiff base deprotonation to the conducting state P510 or, under acidic conditions, to P530
UV-visible Spectroscopic Characterization of ChR2—The UV-visible spectrum of dark-adapted wild type (WT) ChR2 is shown in Fig. 1A
Summary
ChR2 Expression in COS-1 Cells—For expression in COS-1 cells (ATCC, CRL-1650), a human codon-optimized synthetic ChopDNAfragment (corresponding to amino acids 1–311 of the native protein; accession number AF461397), plus the C-terminal ETSQVAPA sequence (1D4 epitope [16]) was designed and purchased from GeneArt (Regensburg, Germany). For experiments at pH 8 and 4.5, freshly prepared ChR2 was diluted with buffer containing 0.03% dodecyl maltoside. For measurements at 80 K, an Oxford Instruments Optistat DN LN2 cooled cryostat was used In this case, the same sample preparation as described below for FTIR measurements was used. A Bruker ifs66v/S FTIR spectrometer with a LN2-cooled mercury cadmium telluride detector (Kolmar Technologies Inc.) and a 1950 cmϪ1 optical cut-off filter was used to record the infrared spectra with a scanning velocity of 200 kHz and a spectral resolution of 2 cmϪ1. A mathematical procedure was developed using the numerical computation language GNU Octave [19] It combines singular value decomposition with a rotation procedure and global fitting to the spectral data as described [20].
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