Localisation of the PsbH subunit in photosystem II: a new approach using labelling of His-tags with a Ni(2+)-NTA gold cluster and single particle analysis.
Photosystem II core dimers were isolated from the green alga Chlamydomonas reinhardtii by Ni(2+)-affinity chromatography exploiting a 6 x His tag located at the N terminus of the PsbH protein. This protein is predicted to have a single transmembrane helix. In order to identify the location of PsbH within the photosystem II complex, the His-tagged core dimers were labelled using a Ni(2+)-NTA gold cluster and subjected to electron microscopy and image analysis. This new method enabled us to identify the location of the labelled His tag by statistical analysis of electron micrographs of the gold-labelled photosystem II complex. Comparison of these data with electron and X-ray crystallographic analysis of photosystem II indicates that the N terminus of PsbH is close to the two transmembrane helices of cytochrome b(559). Our analysis suggests that this approach is a powerful method to locate specific proteins within multisubunit complexes like photosystem II when crystallographic analysis is of insufficient resolution to directly identify amino acid side-chains. Moreover, it can be combined with cross-linking studies, and here we demonstrate that PsbH is a near neighbour of PsbX, which is consistent with the latter subunit being located close to the alpha and beta-subunits of cytochrome b(559). However, cross-linking between PsbH and PsbW was not detected despite the fact that the latter cross-linked with the alpha-subunit of cytochrome b(559).
Büchel
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C
Wolfson Laboratories Department of Biology & Biochemistry, Imperial College of Science, Technology & Medicine, London, SW7 2AY, UK
Morris
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Orlova
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Barber
J
J
D000818:Animals; D015153:Blotting, Western; D016825:Chlamydomonas reinhardtii; D006046:Gold; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods*; D009532:Nickel / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000302:isolation & purification / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D018724:Spinacia oleracea
D008565:Membrane Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C063224:photosystem II, psbH subunit; D006639:Histidine; D006046:Gold; D009532:Nickel