Details

Autor(en) / Beteiligte

• Becker-Baldus, Johanna
• Bamann, Christian
• Saxena, Krishna
• Gustmann, Henrik
• Brown, Lynda J.
• Brown, Richard C. D.
• Reiter, Christian
• Bamberg, Ernst
• Wachtveitl, Josef
• Schwalbe, Harald
• Glaubitz, Clemens

Titel

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 2015-08, Vol.112 (32), p.9896-9901

Ort / Verlag

United States: National Academy of Sciences

Erscheinungsjahr

2015

Quelle

MEDLINE

Beschreibungen/Notizen

• Channelrhodopsin-2 fromChlamydomonas reinhardtiiis 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 retinaltrans-cisisomerization 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 to15N-labeled channelrhodopsin-2 carrying 14,15-13C₂ retinal reconstituted into lipid bilayers. In its dark state, a pure all-transretinal 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 P 1 500 K-like state, the slowly decaying late intermediate P 4 480 , 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.