Site-directed spin-labeling study of the light-harvesting complex CP29.
The topology of the long N-terminal domain (approximately 100 amino-acid residues) of the photosynthetic Lhc CP29 was studied using electron spin resonance. Wild-type protein containing a single cysteine at position 108 and nine single-cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases, the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. The spin-label electron spin resonance spectra were analyzed in terms of a multicomponent spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. These results permit to trace the structural organization of the long N-terminal domain of CP29. Amino-acid residues 97 and 108 are located in the transmembrane pigment-containing protein body of the protein. Positions 65, 81, and 90 are located in a flexible loop that is proposed to extend out of the protein from the stromal surface. This loop also contains a phosphorylation site at Thr81, suggesting that the flexibility of this loop might play a role in the regulatory mechanisms of the light-harvesting process. Positions 4, 33, 40, and 56 are found to be located in a relatively rigid environment, close to the transmembrane protein body. On the other hand, position 15 is located in a flexible region, relatively far away from the transmembrane domain.
Laboratory of Biophysics, Wageningen University, Dreijenlaan 3, NL-6703HA Wageningen, The Netherlands.
Aleh A
AA
Kavalenka
Ruud B
RB
Spruijt
Cor J A M
CJ
Wolfs
Janez
J
Strancar
Roberta
R
Croce
Marcus A
MA
Hemminga
Herbert
H
van Amerongen
D001059:Apoproteins / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002338:Carotenoids / Q000378:metabolism; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D045332:Photosystem II Protein Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D018724:Spinacia oleracea
D001059:Apoproteins; D029681:Arabidopsis Proteins; C069660:CP29 light harvesting complex; D045342:Light-Harvesting Protein Complexes; D045332:Photosystem II Protein Complex; D002338:Carotenoids