Probing the Cl--pumping photocycle of pharaonis halorhodopsin: Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle

Takashi Kikukawa, Chikara Kusakabe, Asami Kokubo, Takashi Tsukamoto, Masakatsu Kamiya, Tomoyasu Aizawa, Kunio Ihara, Naoki Kamo, Makoto Demura

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Halorhodopsin (HR) functions as a light-driven inward Cl- pump. The Cl- transfer process of HR from Natronomonas pharaonis (NpHR) was examined utilizing a mutant strain, KM-1, which expresses large amount of NpHR in a complex with the carotenoid bacterioruberin (Brub). When Cl- was added to unphotolyzed Cl--free NpHR-Brub complex, Brub caused the absorption spectral change in response to the Cl- binding to NpHR through the altered electrostatic environment and/or distortion of its own configuration. During the Cl--puming photocycle, on the other hand, oppositely directed spectral change of Brub appeared during the O intermediate formation and remained until the decay of the last intermediate NpHR′. These results indicate that Cl- is released into the cytoplasmic medium during the N to O transition, and that the subsequent NpHR′ still maintains an altered protein conformation while another Cl- already binds in the vicinity of the Schiff base. Using the cell envelope vesicles, the effect of the interior negative membrane potential on the photocycle was examined. The prominent effect appeared in the shift of the N-O quasi-equilibrium toward N, supporting Cl- release during the N to O transition. The membrane potential had a much larger effect on the Cl- transfer in the cytoplasmic half channel compared to that in the extracellular half channel. This result may reflect the differences in dielectric constants and/or lengths of the pathways for Cl- transfers during N to O and O to NpHR′ transitions.

Original languageEnglish
Pages (from-to)748-758
Number of pages11
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1847
Issue number8
DOIs
Publication statusPublished - May 30 2015

Fingerprint

Halorhodopsins
Membrane Potentials
Coloring Agents
Modulation
Membranes
Protein Conformation
Schiff Bases
Carotenoids
Static Electricity
Conformations
Electrostatics
Permittivity
Pumps
Light
bacterioruberin
Proteins

Keywords

  • Carotenoid
  • Flash photolysis
  • Halorhodopsin
  • Light-driven chloride pump
  • Microbial rhodopsin
  • Photocycle

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Medicine(all)

Cite this

Probing the Cl--pumping photocycle of pharaonis halorhodopsin : Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle. / Kikukawa, Takashi; Kusakabe, Chikara; Kokubo, Asami; Tsukamoto, Takashi; Kamiya, Masakatsu; Aizawa, Tomoyasu; Ihara, Kunio; Kamo, Naoki; Demura, Makoto.

In: Biochimica et Biophysica Acta - Bioenergetics, Vol. 1847, No. 8, 30.05.2015, p. 748-758.

Research output: Contribution to journalArticle

Kikukawa, Takashi ; Kusakabe, Chikara ; Kokubo, Asami ; Tsukamoto, Takashi ; Kamiya, Masakatsu ; Aizawa, Tomoyasu ; Ihara, Kunio ; Kamo, Naoki ; Demura, Makoto. / Probing the Cl--pumping photocycle of pharaonis halorhodopsin : Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle. In: Biochimica et Biophysica Acta - Bioenergetics. 2015 ; Vol. 1847, No. 8. pp. 748-758.
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abstract = "Halorhodopsin (HR) functions as a light-driven inward Cl- pump. The Cl- transfer process of HR from Natronomonas pharaonis (NpHR) was examined utilizing a mutant strain, KM-1, which expresses large amount of NpHR in a complex with the carotenoid bacterioruberin (Brub). When Cl- was added to unphotolyzed Cl--free NpHR-Brub complex, Brub caused the absorption spectral change in response to the Cl- binding to NpHR through the altered electrostatic environment and/or distortion of its own configuration. During the Cl--puming photocycle, on the other hand, oppositely directed spectral change of Brub appeared during the O intermediate formation and remained until the decay of the last intermediate NpHR′. These results indicate that Cl- is released into the cytoplasmic medium during the N to O transition, and that the subsequent NpHR′ still maintains an altered protein conformation while another Cl- already binds in the vicinity of the Schiff base. Using the cell envelope vesicles, the effect of the interior negative membrane potential on the photocycle was examined. The prominent effect appeared in the shift of the N-O quasi-equilibrium toward N, supporting Cl- release during the N to O transition. The membrane potential had a much larger effect on the Cl- transfer in the cytoplasmic half channel compared to that in the extracellular half channel. This result may reflect the differences in dielectric constants and/or lengths of the pathways for Cl- transfers during N to O and O to NpHR′ transitions.",
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T1 - Probing the Cl--pumping photocycle of pharaonis halorhodopsin

T2 - Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle

AU - Kikukawa, Takashi

AU - Kusakabe, Chikara

AU - Kokubo, Asami

AU - Tsukamoto, Takashi

AU - Kamiya, Masakatsu

AU - Aizawa, Tomoyasu

AU - Ihara, Kunio

AU - Kamo, Naoki

AU - Demura, Makoto

PY - 2015/5/30

Y1 - 2015/5/30

N2 - Halorhodopsin (HR) functions as a light-driven inward Cl- pump. The Cl- transfer process of HR from Natronomonas pharaonis (NpHR) was examined utilizing a mutant strain, KM-1, which expresses large amount of NpHR in a complex with the carotenoid bacterioruberin (Brub). When Cl- was added to unphotolyzed Cl--free NpHR-Brub complex, Brub caused the absorption spectral change in response to the Cl- binding to NpHR through the altered electrostatic environment and/or distortion of its own configuration. During the Cl--puming photocycle, on the other hand, oppositely directed spectral change of Brub appeared during the O intermediate formation and remained until the decay of the last intermediate NpHR′. These results indicate that Cl- is released into the cytoplasmic medium during the N to O transition, and that the subsequent NpHR′ still maintains an altered protein conformation while another Cl- already binds in the vicinity of the Schiff base. Using the cell envelope vesicles, the effect of the interior negative membrane potential on the photocycle was examined. The prominent effect appeared in the shift of the N-O quasi-equilibrium toward N, supporting Cl- release during the N to O transition. The membrane potential had a much larger effect on the Cl- transfer in the cytoplasmic half channel compared to that in the extracellular half channel. This result may reflect the differences in dielectric constants and/or lengths of the pathways for Cl- transfers during N to O and O to NpHR′ transitions.

AB - Halorhodopsin (HR) functions as a light-driven inward Cl- pump. The Cl- transfer process of HR from Natronomonas pharaonis (NpHR) was examined utilizing a mutant strain, KM-1, which expresses large amount of NpHR in a complex with the carotenoid bacterioruberin (Brub). When Cl- was added to unphotolyzed Cl--free NpHR-Brub complex, Brub caused the absorption spectral change in response to the Cl- binding to NpHR through the altered electrostatic environment and/or distortion of its own configuration. During the Cl--puming photocycle, on the other hand, oppositely directed spectral change of Brub appeared during the O intermediate formation and remained until the decay of the last intermediate NpHR′. These results indicate that Cl- is released into the cytoplasmic medium during the N to O transition, and that the subsequent NpHR′ still maintains an altered protein conformation while another Cl- already binds in the vicinity of the Schiff base. Using the cell envelope vesicles, the effect of the interior negative membrane potential on the photocycle was examined. The prominent effect appeared in the shift of the N-O quasi-equilibrium toward N, supporting Cl- release during the N to O transition. The membrane potential had a much larger effect on the Cl- transfer in the cytoplasmic half channel compared to that in the extracellular half channel. This result may reflect the differences in dielectric constants and/or lengths of the pathways for Cl- transfers during N to O and O to NpHR′ transitions.

KW - Carotenoid

KW - Flash photolysis

KW - Halorhodopsin

KW - Light-driven chloride pump

KW - Microbial rhodopsin

KW - Photocycle

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