著者

Yuki Sudo Rikou Tanaka Toshitatsu Kobayashi Naoki Kamo Toshiyuki Kohno Chojiro Kojima

出版者

The Biophysical Society of Japan

雑誌

BIOPHYSICS (ISSN:13492942)

巻号頁・発行日

vol.7, pp.51-58, 2011 (Released:2011-06-18)

参考文献数

34

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An approach of cell-free synthesis is presented for the functional expression of transmembrane proteins without the need of refolding. The transmembrane region of the pharaonis halobacterial transducer protein, pHtrII, was translated with various large soluble tags added (thioredoxin, glutathione S-transferase, green fluorescent protein and maltose binding protein). In this system, all fusion pHtrII were translated in a soluble fraction, presumably, forming giant micelle-like structures. The detergent n-dodecyl-β-D-maltoside was added for enhancing the solubilization of the hydrophobic region of pHtrII. The activity of the expressed pHtrII, having various tags, was checked using a pull-down assay, using the fact that pHtrII forms a signaling complex with pharaonis phoborhodopsin (ppR) in the membrane, as also in the presence of a detergent. All tagged pHtrII showed a binding activity with ppR. Interestingly, the binding activity with ppR was positively correlated with the molecular weight of the soluble tags. Thus, larger soluble tags lead to higher binding activities. We could show, that our approach is beneficial for the preparation of active membrane proteins, and is also potentially applicable for larger membrane proteins, such as 7-transmembrane proteins.

著者

Mitsuyuki Kuroki Naoki Kamo Yonosuke Kobatake Yukichi Abe Shin-ichi Ishii

出版者

Japan Society for Cell Biology

雑誌

Cell Structure and Function (ISSN:03867196)

巻号頁・発行日

vol.11, no.2, pp.199-204, 1986 (Released:2008-04-18)

参考文献数

22

被引用文献数

2 2

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Stimulation of polymorphonuclear leukocytes (PMN) by tetravalent concanavalin A (α-ConA) induces membrane depolarization preceding the onset of superoxide anion (O2-) production. Both divalent and monovalent ConA analogues were studied to evaluate the role of valence. Monovalent ConA (m-ConA) was inactive in stimulating O2-production and divalent derivatives were less active than native a-ConA. Similarly, membrane depolarization was dependent on the valency of ConA. m-ConA did not induce a marked change in membrane potential, whereas sustained depolarization occurred with multivalent ConA. The formation of multiple linked interactions between surface receptors may be an important early event in the activation of PMN by ConA.

著者

Jun Tamogami Takashi Kikukawa Toshifumi Nara Makoto Demura Tomomi Kimura-Someya Mikako Shirouzu Shigeyuki Yokoyama Seiji Miyauchi Kazumi Shimono Naoki Kamo

出版者

一般社団法人 日本生物物理学会

雑誌

Biophysics and Physicobiology (ISSN:21894779)

巻号頁・発行日

vol.14, pp.49-55, 2017 (Released:2017-03-18)

参考文献数

25

被引用文献数

7

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A spectrally silent change is often observed in the photocycle of microbial rhodopsins. Here, we suggest the presence of two O intermediates in the photocycle of Acetabularia rhodopsin II (ARII or also called Ace2), a light-driven algal proton pump from Acetabularia acetabulum. ARII exhibits a photocycle including a quasi-equilibrium state of M, N, and O (M<=>N<=>O→) at near neutral and above pH values. However, acidification of the medium below pH ~5.5 causes no accumulation of N, resulting in that the photocycle of ARII can be described as an irreversible scheme (M→O→). This may facilitate the investigation of the latter part of the photocycle, especially the rise and decay of O, during which molecular events have not been sufficiently understood. Thus we analyzed the photocycle under acidic conditions (pH ≤ 5.5). Analysis of the absorbance change at 610 nm, which mainly monitors the fractional concentration changes of K and O, was performed and revealed a photocycle scheme containing two sequential O-states with the different molar extinction coefficients. These photoproducts, termed O1 and O2, may be even produced at physiological pH, although they are not clearly observed under this condition due to the existence of a long M-N-O equilibrium.