High Thermal Stability of Oligomeric Assemblies of Thermophilic Rhodopsin in a Lipid Environment

Tomomi Shionoya, Misao Mizuno, Takashi Tsukamoto, Kento Ikeda, Hayato Seki, Keiichi Kojima, Mikihiro Shibata, Izuru Kawamura, Yuki Sudo, Yasuhisa Mizutani

Research output: Contribution to journalArticle

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Abstract

Thermophilic rhodopsin (TR) is a light-driven proton pump from the extreme thermophile Thermus thermophilus JL-18. Previous studies on TR solubilized with detergent showed that the protein exhibits high thermal stability and forms a trimer at room temperature but irreversibly dissociates into monomers when incubated at physiological temperature (75 °C). In the present study, we used resonance Raman (RR) spectroscopy, solid-state NMR spectroscopy, and high-speed atomic force microscopy to analyze the oligomeric structure of TR in a lipid environment. The obtained spectra and microscopic images demonstrate that TR adopts a pentameric form in a lipid environment and that this assembly is stable at the physiological temperature, in contrast to the behavior of the protein in the solubilized state. These results indicate that the thermal stability of the oligomeric assembly of TR is higher in a lipid environment than in detergent micelles. The observed RR spectra also showed that the retinal chromophore is strongly hydrogen bonded to an internal water molecule via a protonated Schiff base, which is characteristic of proton-pumping rhodopsins. The obtained data strongly suggest that TR functions in the pentameric form at physiological temperature in the extreme thermophile T. thermophilus JL-18. We utilized the high thermal stability of the monomeric form of solubilized TR and here report the first RR spectra of the monomeric form of a microbial rhodopsin. The observed RR spectra revealed that the monomerization of TR alters the chromophore structure: there are changes in the bond alternation of the polyene chain and in the hydrogen-bond strength of the protonated Schiff base. The present study revealed the high thermal stability of oligomeric assemblies of TR in the lipid environment and suggested the importance of using TR embedded in lipid membrane for elucidation of its functional mechanism.

Original languageEnglish
Pages (from-to)6945-6953
Number of pages9
JournalJournal of Physical Chemistry B
Volume122
Issue number27
DOIs
Publication statusPublished - Jul 12 2018

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Rhodopsin
Lipids
assemblies
lipids
Thermodynamic stability
thermal stability
Hot Temperature
Raman scattering
thermophiles
Detergents
Chromophores
detergents
Raman spectra
Protons
imines
chromophores
Proteins
Temperature
assembly
proteins

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry

Cite this

High Thermal Stability of Oligomeric Assemblies of Thermophilic Rhodopsin in a Lipid Environment. / Shionoya, Tomomi; Mizuno, Misao; Tsukamoto, Takashi; Ikeda, Kento; Seki, Hayato; Kojima, Keiichi; Shibata, Mikihiro; Kawamura, Izuru; Sudo, Yuki; Mizutani, Yasuhisa.

In: Journal of Physical Chemistry B, Vol. 122, No. 27, 12.07.2018, p. 6945-6953.

Research output: Contribution to journalArticle

Shionoya, T, Mizuno, M, Tsukamoto, T, Ikeda, K, Seki, H, Kojima, K, Shibata, M, Kawamura, I, Sudo, Y & Mizutani, Y 2018, 'High Thermal Stability of Oligomeric Assemblies of Thermophilic Rhodopsin in a Lipid Environment', Journal of Physical Chemistry B, vol. 122, no. 27, pp. 6945-6953. https://doi.org/10.1021/acs.jpcb.8b04894
Shionoya, Tomomi ; Mizuno, Misao ; Tsukamoto, Takashi ; Ikeda, Kento ; Seki, Hayato ; Kojima, Keiichi ; Shibata, Mikihiro ; Kawamura, Izuru ; Sudo, Yuki ; Mizutani, Yasuhisa. / High Thermal Stability of Oligomeric Assemblies of Thermophilic Rhodopsin in a Lipid Environment. In: Journal of Physical Chemistry B. 2018 ; Vol. 122, No. 27. pp. 6945-6953.
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AU - Shionoya, Tomomi

AU - Mizuno, Misao

AU - Tsukamoto, Takashi

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AU - Seki, Hayato

AU - Kojima, Keiichi

AU - Shibata, Mikihiro

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AU - Sudo, Yuki

AU - Mizutani, Yasuhisa

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AB - Thermophilic rhodopsin (TR) is a light-driven proton pump from the extreme thermophile Thermus thermophilus JL-18. Previous studies on TR solubilized with detergent showed that the protein exhibits high thermal stability and forms a trimer at room temperature but irreversibly dissociates into monomers when incubated at physiological temperature (75 °C). In the present study, we used resonance Raman (RR) spectroscopy, solid-state NMR spectroscopy, and high-speed atomic force microscopy to analyze the oligomeric structure of TR in a lipid environment. The obtained spectra and microscopic images demonstrate that TR adopts a pentameric form in a lipid environment and that this assembly is stable at the physiological temperature, in contrast to the behavior of the protein in the solubilized state. These results indicate that the thermal stability of the oligomeric assembly of TR is higher in a lipid environment than in detergent micelles. The observed RR spectra also showed that the retinal chromophore is strongly hydrogen bonded to an internal water molecule via a protonated Schiff base, which is characteristic of proton-pumping rhodopsins. The obtained data strongly suggest that TR functions in the pentameric form at physiological temperature in the extreme thermophile T. thermophilus JL-18. We utilized the high thermal stability of the monomeric form of solubilized TR and here report the first RR spectra of the monomeric form of a microbial rhodopsin. The observed RR spectra revealed that the monomerization of TR alters the chromophore structure: there are changes in the bond alternation of the polyene chain and in the hydrogen-bond strength of the protonated Schiff base. The present study revealed the high thermal stability of oligomeric assemblies of TR in the lipid environment and suggested the importance of using TR embedded in lipid membrane for elucidation of its functional mechanism.

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