TY - JOUR

T1 - Isolation and Crystallization of the D156C Form of Optogenetic ChR2

AU - Zhang, Liying

AU - Wang, Kaituo

AU - Ning, Shuo

AU - Pedersen, Per Amstrup

AU - Duelli, Annette Susanne

AU - Gourdon, Pontus Emanuel

N1 - Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2022

Y1 - 2022

N2 - 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.

AB - 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.

KW - Channelrhodopsin-2

KW - Crystallization

KW - Open state

KW - Optogenetics

KW - Production

KW - Purification

U2 - 10.3390/cells11050895

DO - 10.3390/cells11050895

M3 - Journal article

C2 - 35269517

AN - SCOPUS:85126008679

VL - 11

JO - Cells

JF - Cells

SN - 2073-4409

IS - 5

M1 - 895

ER -