Two regions of the Mn-stabilizing protein from Synechococcus elongatus that are involved in binding to photosystem II complexes.
Limited proteolysis of the Mn-stabilizing protein (MSP) from the thermophilic cyanobacterium Synechococcus elongatus with chymotrypsin, trypsin or lysylendopeptidase that yielded four major polypeptides of 26 kDa, 22 kDa, 15 kDa and 11 kDa on denaturing gel electrophoresis resulted in total loss of the binding capacity of the protein to PSII complexes. Analyses of electrophoretic patterns and amino-terminal sequences of the proteolytic products revealed that the three proteases specifically cleaved the protein at a site between Phe156 and Gly163 or between Arg184 and Ser191. Site-directed mutagenesis was used to construct two mutant MSPs that had a nick between Phe156 and Leu157, a chymotrypsin-cleavage site, and Met before Leu157 or in place of Leu157. The two mutant proteins failed to bind to PSII complexes, although they largely retained ordered secondary structure and comigrated with the wild-type proteins in non-denaturing gel electrophoresis. The loss of the protein binding can be ascribed to introduction of a nick because a mutant protein that had Met in place of Leu157 but no nick was able to specifically bind to the functional site of PSII complexes and restore the oxygen-evolving activity as effectively as the wild-type protein. In contrast, a mutant MSP with Met inserted between Phe156 and Leu157 bound only weakly and non-specifically to PSII complexes and failed to reactivate oxygen evolution. Thus, the binding of the protein to the functional site of the PSII complex was highly sensitive to a small structural change that was caused by cleavage or insertion of a single amino acid residue between Phe156 and Leu157. The results suggest that the Phe156-Gly163 and Arg184-Ser191 sequences of the cyanobacterial MSP are regions for interaction with PSII complexes.
Motoki
A
A
Biological Sciences Department, Toray Research Center Inc., Kamakura, Japan.
Shimazu
T
T
Hirano
M
M
Katoh
S
S
D000595:Amino Acid Sequence; D000458:Cyanobacteria / Q000737:chemistry* / Q000235:genetics; D010450:Endopeptidases; D008345:Manganese; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D017421:Sequence Analysis
D010446:Peptide Fragments; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D011506:Proteins; C054266:photosystem II manganese-stabilizing protein; D008345:Manganese; D010450:Endopeptidases; D010100:Oxygen