| Autor: |
Zhang L; Department of Biomedical Sciences, University of Copenhagen, Nørre Allé 14, DK-2200 Copenhagen, Denmark., Wang K; Department of Biomedical Sciences, University of Copenhagen, Nørre Allé 14, DK-2200 Copenhagen, Denmark., Ning S; Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, Beijing 100081, China., Pedersen PA; Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark., Duelli AS; Department of Biomedical Sciences, University of Copenhagen, Nørre Allé 14, DK-2200 Copenhagen, Denmark., Gourdon PE; Department of Biomedical Sciences, University of Copenhagen, Nørre Allé 14, DK-2200 Copenhagen, Denmark.; Department of Experimental Medical Science, Lund University, Sölvegatan 19, SE-221 84 Lund, Sweden. |
| Abstrakt: |
Channelrhodopsins (ChRs) are light-gated ion channels that are receiving increasing attention as optogenetic tools. Despite extensive efforts to gain understanding of how these channels function, the molecular events linking light absorption of the retinal cofactor to channel opening remain elusive. While dark-state structures of ChR2 or chimeric proteins have demonstrated the architecture of non-conducting states, there is a need for open- and desensitized-state structures to uncover the mechanistic principles underlying channel activity. To facilitate comprehensive structural studies of ChR2 in non-closed states, we report a production and purification procedure of the D156C form of ChR2, which displays prolonged channel opening compared to the wild type. We demonstrate considerable yields (0.45 mg/g fermenter cell culture) of recombinantly produced protein using S. cerevisiae , which is purified to high homogeneity both as opsin (retinal-free) and as functional ChR2 with added retinal. We also develop conditions that enable the growth of ChR2 crystals that scatter X-rays to 6 Å, and identify a molecular replacement solution that suggests that the packing is different from published structures. Consequently, our cost-effective production and purification pipeline opens the way for downstream structural studies of different ChR2 states, which may provide a foundation for further adaptation of this protein for optogenetic applications. |
