A structural investigation of the central chlorophyll a binding sites in the minor photosystem II antenna protein, Lhcb4.
Mutant proteins from light-harvesting complexes of higher plants may be obtained by expressing modified apoproteins in Escherichia coli, and reconstituting them in the presence of chlorophyll and carotenoid cofactors. This method has allowed, in particular, the engineering of mutant LHCs in which each of the residues coordinating the central Mg atoms of the chlorophylls was replaced by noncoordinating amino acids [Bassi, R., Croce, R., Cugini, D., and Sandonà, D. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10056-10061]. The availability of these mutants is of particular importance for determining the precise position of absorption bands for the different chlorophyll molecules, as well as the sequence of energy transfer events that occur within LHC complexes, provided that the structural impact of each mutation is precisely evaluated. Using resonance Raman spectroscopy, we have characterized the pigment-protein interactions in the minor photosystem II antenna protein, Lhcb4 (CP29), in which each of three of the four central chlorophyll a molecules has been removed by such mutations. By comparing the spectra of these mutants with those of the wild-type protein, the state of interaction of the carbonyl group, the coordination state of the central magnesium ion, and the dielectric constant (polarity) of the immediate environment in the binding pocket of the chlorophyll a molecule were defined for each cofactor binding site. In addition, the structural impact of the absence of one chlorophyll a molecule and the quality of protein folding were evaluated for each of these mutated polypeptides.
Pascal
Andy
A
Università di Verona, Facoltà di Scienze MM.FF.NN., Biotechnologie Vegetali, Strada Le Grazie, I-37134 Verona, Italy. apascal@cea.fr
Caffarri
Stefano
S
Croce
Roberta
R
Sandonà
Dorianna
D
Bassi
Roberto
R
Robert
Bruno
B
D001665:Binding Sites / Q000235:genetics; D002338:Carotenoids / Q000737:chemistry / Q000235:genetics; D002734:Chlorophyll / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000077194:Chlorophyll A; D008024:Ligands; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D011487:Protein Conformation; D017510:Protein Folding; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
C069660:CP29 light harvesting complex; D008024:Ligands; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D002734:Chlorophyll; D002338:Carotenoids; D000077194:Chlorophyll A