A highly active histidine-tagged Chlamydomonas reinhardtii Photosystem II preparation for structural and biophysical analysis.
We describe a genetically engineered strain of Chlamydomonas reinhardtii where the PsbH subunit of Photosystem II (PSII) has been modified to include a C-terminal polyhistidine tag. The strain was generated by the rescue to photoautotrophic growth of a psbH insertional mutant following chloroplast transformation with the modified gene. This selection strategy confirms that the addition of the tag to PsbH does not prevent the assembly of functional PSII, and results in an engineered strain with tagged PSII but no antibiotic-resistance markers in the chloroplast genome. Consequently, the strain is suitable for subsequent genetic manipulation of chloroplast PSII genes. We also describe a rapid PSII isolation procedure that gives a preparation capable of high rates of oxygen evolution. This preparation is suitable for spectroscopic analysis as shown by EPR analysis of the S2 state of the water oxidation cycle. Furthermore, electron microscopy, coupled to single particle analysis, has revealed the isolated PSII to be structurally homogeneous core dimers that are ideally suited for higher resolution structural studies.
Cullen
Matthew
M
Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, South Kensington, London, UK SW7 2AZ.
Ray
Nicola
N
Husain
Sally
S
Nugent
Jonathan
J
Nield
Jon
J
Purton
Saul
S
D020418:Algal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D016825:Chlamydomonas reinhardtii / Q000737:chemistry* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D006639:Histidine / Q000737:chemistry; D046529:Microscopy, Electron, Transmission; D015394:Molecular Structure; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D045332:Photosystem II Protein Complex / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D015202:Protein Engineering; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure
D020418:Algal Proteins; D010750:Phosphoproteins; D045332:Photosystem II Protein Complex; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C063224:photosystem II, psbH subunit; D006639:Histidine