Topological analysis of the extrinsic PsbO, PsbP and PsbQ proteins in a green algal PSII complex by cross-linking with a water-soluble carbodiimide

Ryo Nagao, Takehiro Suzuki, Akinori Okumura, Ayako Niikura, Masako Iwai, Naoshi Dohmae, Tatsuya Tomo, Jian-Ren Shen, Masahiko Ikeuchi, Isao Enami

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

The close association of the extrinsic PsbO, PsbP and PsbQ proteins with PSII core subunits in oxygen-evolving PSII complexes from a green alga, Chlamydomonas reinhardtii, was examined by cross-linking experiments with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The green algal PSII complexes treated with EDC were washed with alkaline Tris to remove the non-cross-linked extrinsic proteins, and then applied to Blue-Native-PAGE to prepare PSII core complexes. The extrinsic proteins cross-linked with PSII core complexes were detected by immunoblotting analysis using antibodies against extrinsic proteins and PSII core subunits. The results showed that the PsbO, PsbP and PsbQ proteins directly associated with CP47, the α subunit of cytochrome b559 and a small subunit in PSII core complexes, respectively, through electrostatic interactions. In addition, a cross-linked product between the PsbP and PsbQ proteins was found in alkaline Tris extracts of EDC-treated PSII complexes, and its cross-linked site was examined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS) after digestions with trypsin and endoproteinase Asp-N. The results demonstrated that the positively charged amino group of K176 on the PsbP protein electrostatically interacts with the negatively charged carboxyl group of D28 on the PsbQ protein. These binding properties of the extrinsic proteins in the green algal PSII were compared with those in higher plant PSII.

Original languageEnglish
Pages (from-to)718-727
Number of pages10
JournalPlant and Cell Physiology
Volume51
Issue number5
DOIs
Publication statusPublished - May 2010

Fingerprint

Carbodiimides
crosslinking
Water
Proteins
proteins
water
endoproteinase Asp-N
Ethyldimethylaminopropyl Carbodiimide
Cytochromes a
oxygen evolving complex
Native Polyacrylamide Gel Electrophoresis
Chlamydomonas reinhardtii
Chlorophyta
electrostatic interactions
matrix-assisted laser desorption-ionization mass spectrometry
binding properties
cytochromes
immunoblotting
Static Electricity
Immunoblotting

Keywords

  • Chlamydomonas reinhardtii
  • Cross-linking
  • Extrinsic protein
  • Oxygen evolution
  • PSII

ASJC Scopus subject areas

  • Plant Science
  • Physiology
  • Cell Biology

Cite this

Topological analysis of the extrinsic PsbO, PsbP and PsbQ proteins in a green algal PSII complex by cross-linking with a water-soluble carbodiimide. / Nagao, Ryo; Suzuki, Takehiro; Okumura, Akinori; Niikura, Ayako; Iwai, Masako; Dohmae, Naoshi; Tomo, Tatsuya; Shen, Jian-Ren; Ikeuchi, Masahiko; Enami, Isao.

In: Plant and Cell Physiology, Vol. 51, No. 5, 05.2010, p. 718-727.

Research output: Contribution to journalArticle

Nagao, Ryo ; Suzuki, Takehiro ; Okumura, Akinori ; Niikura, Ayako ; Iwai, Masako ; Dohmae, Naoshi ; Tomo, Tatsuya ; Shen, Jian-Ren ; Ikeuchi, Masahiko ; Enami, Isao. / Topological analysis of the extrinsic PsbO, PsbP and PsbQ proteins in a green algal PSII complex by cross-linking with a water-soluble carbodiimide. In: Plant and Cell Physiology. 2010 ; Vol. 51, No. 5. pp. 718-727.
@article{07ce4ef1e72d468b92af2e1dcf95d075,
title = "Topological analysis of the extrinsic PsbO, PsbP and PsbQ proteins in a green algal PSII complex by cross-linking with a water-soluble carbodiimide",
abstract = "The close association of the extrinsic PsbO, PsbP and PsbQ proteins with PSII core subunits in oxygen-evolving PSII complexes from a green alga, Chlamydomonas reinhardtii, was examined by cross-linking experiments with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The green algal PSII complexes treated with EDC were washed with alkaline Tris to remove the non-cross-linked extrinsic proteins, and then applied to Blue-Native-PAGE to prepare PSII core complexes. The extrinsic proteins cross-linked with PSII core complexes were detected by immunoblotting analysis using antibodies against extrinsic proteins and PSII core subunits. The results showed that the PsbO, PsbP and PsbQ proteins directly associated with CP47, the α subunit of cytochrome b559 and a small subunit in PSII core complexes, respectively, through electrostatic interactions. In addition, a cross-linked product between the PsbP and PsbQ proteins was found in alkaline Tris extracts of EDC-treated PSII complexes, and its cross-linked site was examined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS) after digestions with trypsin and endoproteinase Asp-N. The results demonstrated that the positively charged amino group of K176 on the PsbP protein electrostatically interacts with the negatively charged carboxyl group of D28 on the PsbQ protein. These binding properties of the extrinsic proteins in the green algal PSII were compared with those in higher plant PSII.",
keywords = "Chlamydomonas reinhardtii, Cross-linking, Extrinsic protein, Oxygen evolution, PSII",
author = "Ryo Nagao and Takehiro Suzuki and Akinori Okumura and Ayako Niikura and Masako Iwai and Naoshi Dohmae and Tatsuya Tomo and Jian-Ren Shen and Masahiko Ikeuchi and Isao Enami",
year = "2010",
month = "5",
doi = "10.1093/pcp/pcq042",
language = "English",
volume = "51",
pages = "718--727",
journal = "Plant and Cell Physiology",
issn = "0032-0781",
publisher = "Oxford University Press",
number = "5",

}

TY - JOUR

T1 - Topological analysis of the extrinsic PsbO, PsbP and PsbQ proteins in a green algal PSII complex by cross-linking with a water-soluble carbodiimide

AU - Nagao, Ryo

AU - Suzuki, Takehiro

AU - Okumura, Akinori

AU - Niikura, Ayako

AU - Iwai, Masako

AU - Dohmae, Naoshi

AU - Tomo, Tatsuya

AU - Shen, Jian-Ren

AU - Ikeuchi, Masahiko

AU - Enami, Isao

PY - 2010/5

Y1 - 2010/5

N2 - The close association of the extrinsic PsbO, PsbP and PsbQ proteins with PSII core subunits in oxygen-evolving PSII complexes from a green alga, Chlamydomonas reinhardtii, was examined by cross-linking experiments with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The green algal PSII complexes treated with EDC were washed with alkaline Tris to remove the non-cross-linked extrinsic proteins, and then applied to Blue-Native-PAGE to prepare PSII core complexes. The extrinsic proteins cross-linked with PSII core complexes were detected by immunoblotting analysis using antibodies against extrinsic proteins and PSII core subunits. The results showed that the PsbO, PsbP and PsbQ proteins directly associated with CP47, the α subunit of cytochrome b559 and a small subunit in PSII core complexes, respectively, through electrostatic interactions. In addition, a cross-linked product between the PsbP and PsbQ proteins was found in alkaline Tris extracts of EDC-treated PSII complexes, and its cross-linked site was examined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS) after digestions with trypsin and endoproteinase Asp-N. The results demonstrated that the positively charged amino group of K176 on the PsbP protein electrostatically interacts with the negatively charged carboxyl group of D28 on the PsbQ protein. These binding properties of the extrinsic proteins in the green algal PSII were compared with those in higher plant PSII.

AB - The close association of the extrinsic PsbO, PsbP and PsbQ proteins with PSII core subunits in oxygen-evolving PSII complexes from a green alga, Chlamydomonas reinhardtii, was examined by cross-linking experiments with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The green algal PSII complexes treated with EDC were washed with alkaline Tris to remove the non-cross-linked extrinsic proteins, and then applied to Blue-Native-PAGE to prepare PSII core complexes. The extrinsic proteins cross-linked with PSII core complexes were detected by immunoblotting analysis using antibodies against extrinsic proteins and PSII core subunits. The results showed that the PsbO, PsbP and PsbQ proteins directly associated with CP47, the α subunit of cytochrome b559 and a small subunit in PSII core complexes, respectively, through electrostatic interactions. In addition, a cross-linked product between the PsbP and PsbQ proteins was found in alkaline Tris extracts of EDC-treated PSII complexes, and its cross-linked site was examined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS) after digestions with trypsin and endoproteinase Asp-N. The results demonstrated that the positively charged amino group of K176 on the PsbP protein electrostatically interacts with the negatively charged carboxyl group of D28 on the PsbQ protein. These binding properties of the extrinsic proteins in the green algal PSII were compared with those in higher plant PSII.

KW - Chlamydomonas reinhardtii

KW - Cross-linking

KW - Extrinsic protein

KW - Oxygen evolution

KW - PSII

UR - http://www.scopus.com/inward/record.url?scp=77952384755&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77952384755&partnerID=8YFLogxK

U2 - 10.1093/pcp/pcq042

DO - 10.1093/pcp/pcq042

M3 - Article

C2 - 20375107

AN - SCOPUS:77952384755

VL - 51

SP - 718

EP - 727

JO - Plant and Cell Physiology

JF - Plant and Cell Physiology

SN - 0032-0781

IS - 5

ER -