Probing the CD lumenal loop region of the D2 protein of photosystem II in Synechocystis sp. strain PCC 6803 by combinatorial mutagenesis

Anna T. Keilty, Svetlana Y. Ermakova-Gerdes, Willem Vermaas

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

The CD lumenal loop region of the photosystem II reaction center protein D2 contains residues involved in oxygen evolution. Since detailed structural information about this region is unavailable, an M13-based combinatorial mutagenesis approach was used to investigate structure-function relationships in this vital region of D2 in Synechocystis sp. strain PCC 6803. The CD loop coding region contains close to 100 nucleotides, and for effective mutagenesis, it was subdivided into four regions of seven to eight codons. A gain-of-function selection protocol was employed such that all mutants that were selected contained a functional D2 protein. In this way, conservation patterns of residues along with numbers and types of amino acid substitutions accommodated at each position for each set of mutants would indicate which residues in the CD loop may play important structural and functional roles. Results of this study have substantiated the importance of residues previously studied by site-directed mutagenesis such as Arg180 and His189 and have identified other previously unremarkable residues in the CD loop (such as Ser166, Phe169, and Ala170) that cannot be replaced by many other residues. In addition, the pliability of the CD loop was further tested using deletion and D1-D2 substitution constructs in M13. This showed that the length of the loop was important to its function, and in two cases, D2 could accommodate homologous sequences from D1, which forms a heterodimer with D2 in photosystem II, but not the other way around. This study of the CD loop in D2 provides valuable clues regarding the structural and functional requirements of the region.

Original languageEnglish (US)
Pages (from-to)2453-2460
Number of pages8
JournalJournal of Bacteriology
Volume182
Issue number9
DOIs
StatePublished - May 2000

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Synechocystis
Photosystem II Protein Complex
Mutagenesis
Amino Acid Substitution
Sequence Homology
Site-Directed Mutagenesis
Codon
Pliability
Proteins
Nucleotides
Oxygen

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

Probing the CD lumenal loop region of the D2 protein of photosystem II in Synechocystis sp. strain PCC 6803 by combinatorial mutagenesis. / Keilty, Anna T.; Ermakova-Gerdes, Svetlana Y.; Vermaas, Willem.

In: Journal of Bacteriology, Vol. 182, No. 9, 05.2000, p. 2453-2460.

Research output: Contribution to journalArticle

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