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

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 MU-based combinatorial mut agenesis approach was used to investigate structure-function relationships in this vital region of D2 in Synechocystis sp, strain PCC 6803. The CD loo p coding region contains close to 100 nucleotides, and for effective mutage nesis, it was subdivided into four regions of seven to eight codons. A gain -of-function selection protocol was employed such that all mutants that,wer e selected contained a functional D2 protein. In this way, conservation pat terns 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 previo usly studied by site-directed mutagenesis such as Arg180 and His189 and hav e 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 delet ion and D1-D2 substitution constructs in M13. This showed that the length o f the loop was important to its function, and in two cases, D2 could accomm odate homologous sequences from D1, which forms a heterodimer dth D2 in pho tosystem II, but not the other way around. This study of the CD loop in D2 provides valuable clues regarding the structural and functional requirement s of the region.