Journal Article

Identification of Functional Domains of the Extrinsic 12 kDa Protein in Red Algal PSII by Limited Proteolysis and Directed Mutagenesis

Akinori Okumura, Hisataka Ohta, Yasunori Inoue and Isao Enami

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 42, issue 12, pages 1331-1337
Published in print December 2001 | ISSN: 0032-0781
Published online December 2001 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pce170
Identification of Functional Domains of the Extrinsic 12 kDa Protein in Red Algal PSII by Limited Proteolysis and Directed Mutagenesis

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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The extrinsic 12 kDa protein in red algal photosystem II (PSII) functions to minimize the chloride and calcium requirement of oxygen-evolving activity [Enami et al. (1998) Biochemistry 37: 2787]. In order to identify functional domains of the 12 kDa protein, we prepared the 12 kDa protein lacking N-terminal peptides or C-terminal peptides or both by limited proteolysis and directed mutagenesis. The resulting 12 kDa protein fragments were examined for their binding and functional properties by reconstitution experiments. (1) A peptide fragment from Gly-6 to C-terminus of the 12 kDa protein was prepared by V8 protease. This fragment rebound to PSII completely, and it reactivated oxygen evolution partially in the absence of Cl and Ca2+ ions but significantly in the presence of Cl ion. (2) A peptide from Leu-10 to Phe-83 was obtained by chymotrypsin treatment. This peptide rebound to PSII effectively, but the rebinding did not restore oxygen evolution in both the absence and presence of Cl and Ca2+ ions. (3) Two mutant proteins, one lacking five residues and the other lacking nine residues of the N-terminus, were able to bind to PSII effectively. Recovery of oxygen evolution by their binding was almost the same as that reconstituted with the V8 protease-treated peptide. (4) Three mutant proteins lacking ten, seven or three residues of the C-terminus effectively rebound to PSII, but their binding did not result in recovery of the oxygen evolution. In contrast, reconstitution with a mutant protein lacking one residue of the C-terminus showed the same high restoration of oxygen evolution as reconstitution with the full-length 12 kDa protein. (5) These results indicate that two residues from lysine of the C-terminus of the 12 kDa protein constitute an important domain for minimizing the chloride and calcium requirement of oxygen evolution. In addition, the N-terminus of the protein, at least five residues, has a secondary function for the chloride requirement.

Keywords: Key words: Extrinsic 12 kDa protein — Directed mutagenesis — Functional domains — Limited proteolysis — Oxygen-evolving PSII — Red algae.; Abbreviations: CP, chlorophyll-binding protein; LIC, ligation independent cloning; PVDF, polyvinylidene fluoride.

Journal Article.  4592 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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