TY - JOUR
T1 - Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization
AU - Kikuchi, Masuzu
AU - Kojima, Keiichi
AU - Nakao, Shin
AU - Yoshizawa, Susumu
AU - Kawanishi, Shiho
AU - Shibukawa, Atsushi
AU - Kikukawa, Takashi
AU - Sudo, Yuki
N1 - Funding Information:
We wish to thank Drs. Masayuki Sakamoto and Haruhiko Bito for providing the expression plasmid of OmR2 for mammalian cells. This work was financially supported by JSPS KAKENHI Grant Numbers JP19K16090 to KK, JP18H04136 to SY, JP17K07326 to TK, JP19H04727, JP19H05396, JP20K21482, JP21H0040413, JP18H02411, and JP21H0244613 to YS. This research was partially supported by JST CREST (JPMJCR1656) and AMED (20dm0207060h0004) to YS. The authors also thank “DASS Manuscript” (http://www.dass-ms.com/home.html) for the English language review.
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12
Y1 - 2021/12
N2 - Microbial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.
AB - Microbial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.
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U2 - 10.1038/s41598-021-94181-w
DO - 10.1038/s41598-021-94181-w
M3 - Article
C2 - 34285294
AN - SCOPUS:85111106171
VL - 11
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
IS - 1
M1 - 14765
ER -