Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants.
A chimeric Lhcb gene encoding light-harvesting chlorophyll a/b-binding protein (LHCII) was expressed in transgenic tobacco plants. To separate native from recombinant LHCII, the protein was extended by six histidines at its C terminus. Recombinant LHCII was isolated by detergent-mediated monomerization of pure trimers followed by affinity-chromatography on Ni(2+)-NTA-agarose (NTA is nitrilotriacetic acid). Elution with imidazole yielded recombinant monomers that formed trimers readily after dilution of the detergent without further in vitro manipulations. LHCII subunits showed the typical chlorophyll a/b ratio at all steps of purification indicating no significant loss of pigments. Transgenic tobacco overexpressed amounts of recombinant protein that corresponded to about 0.7% of total LHCII. This yield suggested that expression in planta might be an alternative to the expression of eukaryotic membrane proteins in yeast. Recombinant LHCII was able to form two-dimensional crystals after addition of digalactolipids, which diffracted electrons to 3.6-A resolution. LHCII carrying a replacement of Arg-21 with Gln accumulated to only 0.004% of total thylakoid proteins. This mutant was monomeric in the photosynthetic membrane probably due to the deletion of the phosphatidylglycerol binding site and was degraded by the plastidic proteolytic system. Exchange of Asn-183 with Leu impaired LHCII biogenesis in a similar way presumably due to the lack of a chlorophyll a binding site.
Flachmann
R
R
European Molecular Biology Laboratory, Heidelberg, Germany.
Kühlbrandt
W
W
D029681:Arabidopsis Proteins; D001483:Base Sequence; D061146:Chlorophyll Binding Proteins; D003460:Crystallization; D017343:Genes, Plant; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000235:genetics*; D045331:Photosystem I Protein Complex; D010940:Plant Proteins; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*
D029681:Arabidopsis Proteins; D061146:Chlorophyll Binding Proteins; C490278:LHCA1 protein, Arabidopsis; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D010940:Plant Proteins; D011994:Recombinant Proteins