Isolation and Crystallization of the D156C Form of Optogenetic ChR2

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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.

Udgave nummer5
Antal sider11
StatusUdgivet - 2022

Bibliografisk note

Funding Information:
Funding: L.Z. was supported by a PhD scholarship from the China Scholarship Council (CSC). A.S.D. was supported by postdoctoral scholarships from The William Harvey International Translational Research Academy. P.A.P. was supported by the Independent Research Fund Denmark, the Novo-Nordic Foundation, and the Villum Foundation. P.E.G. was financed by the following Foundations: Lundbeck, Knut and Alice Wallenberg, Carlsberg, Novo-Nordisk, Brødrene Hartmann, Agnes og Poul Friis, Augustinus, and Crafoord as well as Per-Eric and Ulla Schyberg. Funding was also obtained from the Independent Research Fund Denmark, the Swedish Research Council, and through a Michaelsen scholarship.

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© 2022 by the authors. Licensee MDPI, Basel, Switzerland.

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