Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy
Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy
Channelrhodopsin-2 from Chlamydomonas reinhardtii is a light-gated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to 15N-labeled channelrhodopsin-2 carrying 14,15-13C2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early P5001 K-like state, the slowly decaying late intermediate P4804, and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray–based structure analysis, which is required for resolving molecular mechanisms.
9896-9901
Becker-Baldus, Johanna
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Bamann, Christian
332a26c0-befa-43b0-a3b6-efa344cf644c
Saxena, Krishna
67ca0436-c799-406c-a6e3-704ea299077c
Gustmann, Henrik
cc89d60e-8849-4397-9a80-279e0ba217f0
Brown, Lynda J.
75aa95fa-5d27-46a7-9dbe-0f465a664f5b
Brown, Richard C.D.
21ce697a-7c3a-480e-919f-429a3d8550f5
Reiter, Christian
444c22b5-2b1d-49fb-aaa7-0428562dc7ed
Bamberg, Ernst
afff58bc-8040-4061-9b56-5f020496e726
Wachtveitl, Josef
b33cef4d-67a0-4804-b0f7-90c408a33a05
Schwalbe, Harald
66e728b1-14e2-4e31-ab27-427a52dd3b4d
Glaubitz, Clemens
99f5e847-e6fd-4783-bc60-054bf0e15661
11 August 2015
Becker-Baldus, Johanna
6563d44b-d7aa-4e93-b7c2-57b95f8a0f1d
Bamann, Christian
332a26c0-befa-43b0-a3b6-efa344cf644c
Saxena, Krishna
67ca0436-c799-406c-a6e3-704ea299077c
Gustmann, Henrik
cc89d60e-8849-4397-9a80-279e0ba217f0
Brown, Lynda J.
75aa95fa-5d27-46a7-9dbe-0f465a664f5b
Brown, Richard C.D.
21ce697a-7c3a-480e-919f-429a3d8550f5
Reiter, Christian
444c22b5-2b1d-49fb-aaa7-0428562dc7ed
Bamberg, Ernst
afff58bc-8040-4061-9b56-5f020496e726
Wachtveitl, Josef
b33cef4d-67a0-4804-b0f7-90c408a33a05
Schwalbe, Harald
66e728b1-14e2-4e31-ab27-427a52dd3b4d
Glaubitz, Clemens
99f5e847-e6fd-4783-bc60-054bf0e15661
Becker-Baldus, Johanna, Bamann, Christian, Saxena, Krishna, Gustmann, Henrik, Brown, Lynda J., Brown, Richard C.D., Reiter, Christian, Bamberg, Ernst, Wachtveitl, Josef, Schwalbe, Harald and Glaubitz, Clemens
(2015)
Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy.
Proceedings of the National Academy of Sciences, 112 (32), .
(doi:10.1073/pnas.1507713112).
(PMID:26216996)
Abstract
Channelrhodopsin-2 from Chlamydomonas reinhardtii is a light-gated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to 15N-labeled channelrhodopsin-2 carrying 14,15-13C2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early P5001 K-like state, the slowly decaying late intermediate P4804, and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray–based structure analysis, which is required for resolving molecular mechanisms.
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More information
Accepted/In Press date: 25 June 2015
e-pub ahead of print date: 27 July 2015
Published date: 11 August 2015
Additional Information:
Channelrhodopsin-2 is a dimeric membrane protein functioning as a light-gated ion channel, which has triggered numerous optogenetic applications. We present the first NMR study, to our knowledge, by which structural details of the retinal cofactor could be resolved. This study was only possible by enhancing the detection sensitivity 60-fold through dynamic nuclear polarization (DNP), a highly promising hybrid method linking EPR with solid-state NMR spectroscopy. Our data show that ground-state channelrhodopsin-2 contains the retinal cofactor in its all-trans configuration with a slightly perturbed polyene chain. Three different photointermediates could be trapped and analyzed. Our study shows that DNP-enhanced solid-state NMR is a key method for bridging the gap between X-ray–based structure analysis and functional studies toward a highly resolved molecular picture.
Organisations:
Organic Chemistry: Synthesis, Catalysis and Flow
Identifiers
Local EPrints ID: 383950
URI: http://eprints.soton.ac.uk/id/eprint/383950
ISSN: 0027-8424
PURE UUID: 01870617-f92f-4567-837f-6338a851e702
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Date deposited: 30 Nov 2015 14:02
Last modified: 15 Mar 2024 02:59
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Contributors
Author:
Johanna Becker-Baldus
Author:
Christian Bamann
Author:
Krishna Saxena
Author:
Henrik Gustmann
Author:
Christian Reiter
Author:
Ernst Bamberg
Author:
Josef Wachtveitl
Author:
Harald Schwalbe
Author:
Clemens Glaubitz
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