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PMID: 18927082 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Monitoring light-induced structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform infrared spectroscopy.

The Journal of biological chemistry ·Vol. 283 ·No. 50 ·2008-12-12 ·Pages 35033-41

Ritter E, Stehfest K, Berndt A, Hegemann P, Bartl FJ

Abstract

Channelrhodopsin-2 (ChR2) is a microbial type rhodopsin and a light-gated cation channel that controls phototaxis in Chlamydomonas. We expressed ChR2 in COS-cells, purified it, and subsequently investigated this unusual photoreceptor by flash photolysis and UV-visible and Fourier transform infrared difference spectroscopy. Several transient photoproducts of the wild type ChR2 were identified, and their kinetics and molecular properties were compared with those of the ChR2 mutant E90Q. Based on the spectroscopic data we developed a model of the photocycle comprising six distinguishable intermediates. This photocycle shows similarities to the photocycle of the ChR2-related Channelrhodopsin of Volvox but also displays significant differences. We show that molecular changes include retinal isomerization, changes in hydrogen bonding of carboxylic acids, and large alterations of the protein backbone structure. These alterations are stronger than those observed in the photocycle of other microbial rhodopsins like bacteriorhodopsin and are related to those occurring in animal rhodopsins. UV-visible and Fourier transform infrared difference spectroscopy revealed two late intermediates with different time constants of tau = 6 and 40 s that exist during the recovery of the dark state. The carboxylic side chain of Glu(90) is involved in the slow transition. The molecular changes during the ChR2 photocycle are discussed with respect to other members of the rhodopsin family.

MeSH Terms
Animals COS Cells Chlamydomonas/metabolism Chlorocebus aethiops Hydrogen Bonding Hydrogen-Ion Concentration Kinetics Light Protein Structure, Tertiary Proteins/chemistry Rhodopsin/chemistry Spectrophotometry, Ultraviolet/methods Spectroscopy, Fourier Transform Infrared/methods Time Factors Ultraviolet Rays
Chemicals
Proteins Rhodopsin
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Ritter Eglof
Institut für medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
Stehfest Katja
Berndt Andre
Hegemann Peter
Bartl Franz J
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Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
0021-9258
DOI
10.1074/jbc.M806353200
Published
2008-12-12
Epub
2008-00-16
Pages
35033-41
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC3259888
Subset
IM
External Links
PubMed source DOI: 10.1074/jbc.M806353200 PMC Full Text View journal details
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