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Makino, Yoshiteru Graduate School of Engineering, Yokohama National University
Kawamura, Izuru Graduate School of Engineering, Yokohama National University
Okitsu, Takashi Laboratory of Organic Chemistry for Life Science, Kobe Pharmaceutical University
Wada, Akimori Laboratory of Organic Chemistry for Life Science, Kobe Pharmaceutical University
Kamo, Naoki Faculty of Advanced Life Science, Hokkaido University
Sudo, Yuki Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University
Ueda, Kazuyoshi Graduate School of Engineering, Yokohama National University
Naito, Akira Graduate School of Engineering, Yokohama National University
Abstract
Pharanois phoborhodopsin (ppR) from Natronomonas pharaonis is a transmembrane photoreceptor protein involved in negative phototaxis. Structural changes in ppR triggered by photoisomerization of the retinal chromophore are transmitted to its cognate transducer protein (pHtrII) through a cyclic photoreaction pathway involving several photointermediates. This pathway is called the photocycle. It is important to understand the detailed configurational changes of retinal during the photocycle. We previously observed one of the photointermediates (M-intermediates) by in situ photoirradiation solid-state NMR experiments. In this study, we further observed the 13C cross-polarization magic-angle-spinning NMR signals of late photointermediates such as O- and N'-intermediates by illumination with green light (520 nm). Under blue-light (365 nm) irradiation of the M-intermediates, 13C cross-polarization magic-angle-spinning NMR signals of 14- and 20-13C-labeled retinal in the O-intermediate appeared at 115.4 and 16.4 ppm and were assigned to the 13-trans, 15-syn configuration. The signals caused by the N'-intermediate appeared at 115.4 and 23.9 ppm and were assigned to the 13-cis configuration, and they were in an equilibrium state with the O-intermediate during thermal decay of the M-intermediates at -60°C. Thus, photoirradiation NMR studies revealed the photoreaction pathways from the M- to O-intermediates and the equilibrium state between the N'- and O-intermediate. Further, we evaluated the detailed retinal configurations in the O- and N'-intermediates by performing a density functional theory chemical shift calculation. The results showed that the N'-intermediate has a 63° twisted retinal state due to the 13-cis configuration. The retinal configurations of the O- and N'-intermediates were determined to be 13-trans, 15-syn, and 13-cis, respectively, based on the chemical shift values of [20-13C] and [14-13C] retinal obtained by photoirradiation solid-state NMR and density functional theory calculation.
Note
This is an Accepted Manuscript of an article published by Biophysical Society
This fulltext will be available in Jul 2019
Published Date
2018-07-03
Publication Title
Biophysical Journal
Volume
volume115
Issue
issue1
Publisher
Biophysical Society
Start Page
72
End Page
83
ISSN
00063495
NCID
AA00566095
Content Type
Journal Article
language
英語
OAI-PMH Set
岡山大学
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isVersionOf https://doi.org/10.1016/j.bpj.2018.05.030