Transient movement of helix F revealed by photo-induced inactivation by reaction of a bulky SH-reagent to cysteine-introduced pharaonis phoborhodopsin (sensory rhodopsin II)

Hideaki Yoshida, Yuki Sudo, Kazumi Shimono, Masayuki Iwamoto, Naoki Kamo

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Pharaonis phoborhodopsin (ppR) is a photosensor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis, an alkalophilic halophile. This protein has seven transmembrane helices into which a chromophore, all-trans retinal, binds to a specific lysine residue (located in helix G) via a protonated Schiff base. Various mutants were engineered to have a single cysteine in the F-helix. In the presence of a bulky fluorescent SH-reagent, MIANS, (2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid, illumination decreased the photoreactivity or flash-yield (absorbance deflection immediately after the flash) of the L163C ppR mutant (in which Leu-163 was replaced with Cys) without changing the photocycling rate. The fluorescence of the isolated protein increased with increasing illumination. These observations suggest that during photocycling, the space around Cys-163 in the F-helix might open, permitting reaction with the relatively large molecule. This reaction occurred only at the M-state and not at the O-state. The implications are discussed.

Original languageEnglish
Pages (from-to)537-542
Number of pages6
JournalPhotochemical and Photobiological Sciences
Volume3
Issue number6
DOIs
Publication statusPublished - Jun 2004
Externally publishedYes

Fingerprint

Sensory Rhodopsins
Sulfhydryl Reagents
cysteine
Lighting
deactivation
helices
Cysteine
reagents
Natronobacterium
Photoreactivity
Schiff Bases
Chromophores
Lysine
halophiles
flash
Proteins
Fluorescence
illumination
photosensors
proteins

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Cell Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biophysics

Cite this

Transient movement of helix F revealed by photo-induced inactivation by reaction of a bulky SH-reagent to cysteine-introduced pharaonis phoborhodopsin (sensory rhodopsin II). / Yoshida, Hideaki; Sudo, Yuki; Shimono, Kazumi; Iwamoto, Masayuki; Kamo, Naoki.

In: Photochemical and Photobiological Sciences, Vol. 3, No. 6, 06.2004, p. 537-542.

Research output: Contribution to journalArticle

@article{68f2e8ad02dd4c65af84cba532349a1c,
title = "Transient movement of helix F revealed by photo-induced inactivation by reaction of a bulky SH-reagent to cysteine-introduced pharaonis phoborhodopsin (sensory rhodopsin II)",
abstract = "Pharaonis phoborhodopsin (ppR) is a photosensor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis, an alkalophilic halophile. This protein has seven transmembrane helices into which a chromophore, all-trans retinal, binds to a specific lysine residue (located in helix G) via a protonated Schiff base. Various mutants were engineered to have a single cysteine in the F-helix. In the presence of a bulky fluorescent SH-reagent, MIANS, (2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid, illumination decreased the photoreactivity or flash-yield (absorbance deflection immediately after the flash) of the L163C ppR mutant (in which Leu-163 was replaced with Cys) without changing the photocycling rate. The fluorescence of the isolated protein increased with increasing illumination. These observations suggest that during photocycling, the space around Cys-163 in the F-helix might open, permitting reaction with the relatively large molecule. This reaction occurred only at the M-state and not at the O-state. The implications are discussed.",
author = "Hideaki Yoshida and Yuki Sudo and Kazumi Shimono and Masayuki Iwamoto and Naoki Kamo",
year = "2004",
month = "6",
doi = "10.1039/b315454h",
language = "English",
volume = "3",
pages = "537--542",
journal = "Photochemical and Photobiological Sciences",
issn = "1474-905X",
publisher = "Royal Society of Chemistry",
number = "6",

}

TY - JOUR

T1 - Transient movement of helix F revealed by photo-induced inactivation by reaction of a bulky SH-reagent to cysteine-introduced pharaonis phoborhodopsin (sensory rhodopsin II)

AU - Yoshida, Hideaki

AU - Sudo, Yuki

AU - Shimono, Kazumi

AU - Iwamoto, Masayuki

AU - Kamo, Naoki

PY - 2004/6

Y1 - 2004/6

N2 - Pharaonis phoborhodopsin (ppR) is a photosensor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis, an alkalophilic halophile. This protein has seven transmembrane helices into which a chromophore, all-trans retinal, binds to a specific lysine residue (located in helix G) via a protonated Schiff base. Various mutants were engineered to have a single cysteine in the F-helix. In the presence of a bulky fluorescent SH-reagent, MIANS, (2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid, illumination decreased the photoreactivity or flash-yield (absorbance deflection immediately after the flash) of the L163C ppR mutant (in which Leu-163 was replaced with Cys) without changing the photocycling rate. The fluorescence of the isolated protein increased with increasing illumination. These observations suggest that during photocycling, the space around Cys-163 in the F-helix might open, permitting reaction with the relatively large molecule. This reaction occurred only at the M-state and not at the O-state. The implications are discussed.

AB - Pharaonis phoborhodopsin (ppR) is a photosensor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis, an alkalophilic halophile. This protein has seven transmembrane helices into which a chromophore, all-trans retinal, binds to a specific lysine residue (located in helix G) via a protonated Schiff base. Various mutants were engineered to have a single cysteine in the F-helix. In the presence of a bulky fluorescent SH-reagent, MIANS, (2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid, illumination decreased the photoreactivity or flash-yield (absorbance deflection immediately after the flash) of the L163C ppR mutant (in which Leu-163 was replaced with Cys) without changing the photocycling rate. The fluorescence of the isolated protein increased with increasing illumination. These observations suggest that during photocycling, the space around Cys-163 in the F-helix might open, permitting reaction with the relatively large molecule. This reaction occurred only at the M-state and not at the O-state. The implications are discussed.

UR - http://www.scopus.com/inward/record.url?scp=3242746807&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=3242746807&partnerID=8YFLogxK

U2 - 10.1039/b315454h

DO - 10.1039/b315454h

M3 - Article

C2 - 15170482

AN - SCOPUS:3242746807

VL - 3

SP - 537

EP - 542

JO - Photochemical and Photobiological Sciences

JF - Photochemical and Photobiological Sciences

SN - 1474-905X

IS - 6

ER -