In vitro reconstitution of a light-harvesting gene product: deletion mutagenesis and analyses of pigment binding.
AB96, a gene encoding a Pisum sativum chlorophyll a/b binding protein [Coruzzi et al. (1983) J. Biol. Chem. 258, 1399-1402], can be expressed in Escherichia coli and reconstituted with pigments by the procedure described by Plumley and Schmidt [(1987) Proc. Natl. Acad. Sci. U.S.A. 84, 146-150]. Following purification by polyacrylamide gel electrophoresis, the reconstituted pigment-protein complex (CP2) is shown to have similar pigment-binding characteristics to native CP2 complexes isolated from thylakoid membranes. Therefore, the AB96 gene product contains binding sites for chlorophylls a and b and xanthophylls, all of which are necessary for optimal reconstitution in vitro. Absorption, fluorescence, and circular dichroism spectroscopy indicate that the pigments are oriented accurately and that chlorophylls a and b are adjoined for energy transfer. Studies with proteins produced after deletion mutagenesis of AB96 indicate that NH2-terminal amino acids 1-21 and COOH-terminal amino acids 219-228 do not play a role in pigment binding. In contrast, amino acids 50-57 and 204-212 (encompassing one of three conserved histidine residues) are essential for reconstitution. Residues near the presumed NH2- and COOH-terminal alpha-helix boundaries (22-49 and 213-218, respectively) affect the stability of reconstituted CP2 during electrophoresis at 4 degrees C. Correlation of diminished chlorophyll a binding with disappearance of a negative circular dichroism near 684 nm suggests that amino acids 213-218 near the COOH-terminal boundary of the third membrane-spanning helix affect the binding of some chlorophyll a molecules.
Cammarata
K V
KV
Botany Department, University of Georgia, Athens 30602.
Schmidt
G W
GW
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002872:Chromosome Deletion; D002942:Circular Dichroism; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007887:Fabaceae / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D007425:Intracellular Membranes / Q000378:metabolism; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D016296:Mutagenesis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics* / Q000378:metabolism; D010860:Pigments, Biological / Q000378:metabolism*; D010946:Plants, Medicinal; D013050:Spectrometry, Fluorescence
D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D010860:Pigments, Biological; D004247:DNA