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Isolation and Crystallization of the D156C Form of Optogenetic ChR2

Zhang, Liying LU ; Wang, Kaituo ; Ning, Shuo ; Pedersen, Per Amstrup ; Duelli, Annette Susanne and Gourdon, Pontus Emanuel LU (2022) In Cells 11(5).
Abstract

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... (More)

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.

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author
; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Channelrhodopsin-2, Crystallization, Open state, Optogenetics, Production, Purification
in
Cells
volume
11
issue
5
article number
895
publisher
MDPI AG
external identifiers
  • pmid:35269517
  • scopus:85126008679
ISSN
2073-4409
DOI
10.3390/cells11050895
language
English
LU publication?
yes
id
fdf6ea81-9e26-4310-b02b-8645865a4fa5
date added to LUP
2022-04-26 11:30:56
date last changed
2024-06-27 13:15:56
@article{fdf6ea81-9e26-4310-b02b-8645865a4fa5,
  abstract     = {{<p>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.</p>}},
  author       = {{Zhang, Liying and Wang, Kaituo and Ning, Shuo and Pedersen, Per Amstrup and Duelli, Annette Susanne and Gourdon, Pontus Emanuel}},
  issn         = {{2073-4409}},
  keywords     = {{Channelrhodopsin-2; Crystallization; Open state; Optogenetics; Production; Purification}},
  language     = {{eng}},
  month        = {{03}},
  number       = {{5}},
  publisher    = {{MDPI AG}},
  series       = {{Cells}},
  title        = {{Isolation and Crystallization of the D156C Form of Optogenetic ChR2}},
  url          = {{http://dx.doi.org/10.3390/cells11050895}},
  doi          = {{10.3390/cells11050895}},
  volume       = {{11}},
  year         = {{2022}},
}