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Proc Natl Acad Sci U S A. 2015 Aug 11;112(32):9896-901. doi: 10.1073/pnas.1507713112. Epub 2015 Jul 27.

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

Author information

1
Institute of Biophysical Chemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany; Centre for Biomolecular Magnetic Resonance, Goethe University Frankfurt, 60438 Frankfurt, Germany;
2
Max-Planck-Institute of Biophysics, 60438 Frankfurt, Germany;
3
Centre for Biomolecular Magnetic Resonance, Goethe University Frankfurt, 60438 Frankfurt, Germany; Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt, 60438 Frankfurt, Germany;
4
Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany;
5
Department of Chemistry, University of Southampton, Southampton SO17 1BJ, United Kingdom;
6
Bruker Biospin GmbH, 76287 Rheinstetten, Germany.
7
Institute of Biophysical Chemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany; Centre for Biomolecular Magnetic Resonance, Goethe University Frankfurt, 60438 Frankfurt, Germany; glaubitz@em.uni-frankfurt.de.

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 (15)N-labeled channelrhodopsin-2 carrying 14,15-(13)C2 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 [Formula: see text] K-like state, the slowly decaying late intermediate [Formula: see text], 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.

KEYWORDS:

DNP; channelrhodopsin; freeze trapping; retinal; solid-state NMR

PMID:
26216996
PMCID:
PMC4538646
DOI:
10.1073/pnas.1507713112
[Indexed for MEDLINE]
Free PMC Article

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