Statische und zeitaufgelöste FTIR Differenzspektroskopie an Kanalrhodopsinen aus Chlamydomonas reinhardtii und Volvox carteri
Final Report Abstract
In the project “Statische und zeitaufgelöste FTIR Differenzspektroskopie an Kanalrhodopsinen aus Chlamydomonas reinhardtii und Volvox carteri“ we investigated the activation mechanism of channelrhodopsin-2 by UV/ Vis and FTIR spectroscopy as well as by electrical measurements and MD simulations. We focused on mutants of the residues C128 and E90. Mutants of C128 are of special interest since they significantly slow down the photocycle which makes it extremely useful for applications in neurosciences. E90 is interesting because it acts as a selectivity filter for different ions and therefore gives insight into to the mechanism of channel formation and ion conductance. We could show that during a late stage of the C128 photocycle, a fraction of the protein reacts into a side branch involving an inactive species P380, which accumulates during prolonged illumination. At neutral pH P380 evolves into P353 which contains the chromophore in a retroretinyl configuration as indicated by its characteristic fine structured UV/ Vis spectrum. Furthermore, for the C128T mutant three different dark states were identified whose formation strongly depend on the illumination conditions. By evaluating the time resolved FTIR data sets by a combination of singular value decomposition and global analysis we could show that the activation mechanism of ChR2 involves two distinct mechanistic steps: First, formation of a pre-conducting that is still blocked and non-conducting, linked to large structural changes, and second, the “gating”, in which the block is removed leading to the conducting pore This step requires only small structural changes. Based on the UV/ Vis and FTIR data and on data obtained by HPLC analysis we developed a reaction model for C128T, with two interconnected photo cycles, involving the three dark states and starting from an all-trans, 15-anti and 13-cis,15 syn retinal conformation. Time resolved FTIR spectroscopy revealed that E90 becomes deprotonated during the photo cycle. Global analysis of the data set revealed that that this deprotonation occurs before formation of the conducting state P520 and is reversed in the last step of the photo cycle during dark state recovery. Similar observations were made for secondary structural changes of the receptor during the photocycle.
Publications
- “The branched photocycle of the slow-cycling mutant C128T”, J. Mol. Biol., (2010), 398, 690-702
K. Stehfest, E. Ritter, A. Berndt, F. Bartl and P. Hegemann
- “In Channelrhodopsin-2 Glu-90 is crucial for ion selectivity and is deprotonated during the photocycle, J. Biol. Chem., (2012), 287, 6904 - 6911
K. Eisenhauer, J. Kuhne, E., Ritter, A. Bernst, S. Wolff, E. Freier, F. Bartl, P. Hegemann and K. Gerwert
(See online at https://doi.org/10.1074/jbc.M111.327700) - “Light-dark adaptation of channelrhodopsin C128T mutant, J Biol Chem. (2013), 288, 10451 - 10458
E. Ritter, P. Piwowarski, P. Hegemann and F. Bartl
(See online at https://doi.org/10.1074/jbc.M112.446427)
