| Abstract
| - Mutant proteins from light-harvesting complexes of higher plants may be obtained by expressingmodified apoproteins in Escherichiacoli, and reconstituting them in the presence of chlorophyll andcarotenoid cofactors. This method has allowed, in particular, the engineering of mutant LHCs in whicheach of the residues coordinating the central Mg atoms of the chlorophylls was replaced by noncoordinatingamino 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 preciseposition of absorption bands for the different chlorophyll molecules, as well as the sequence of energytransfer events that occur within LHC complexes, provided that the structural impact of each mutation isprecisely evaluated. Using resonance Raman spectroscopy, we have characterized the pigment−proteininteractions in the minor photosystem II antenna protein, Lhcb4 (CP29), in which each of three of thefour central chlorophyll a molecules has been removed by such mutations. By comparing the spectra ofthese mutants with those of the wild-type protein, the state of interaction of the carbonyl group, thecoordination state of the central magnesium ion, and the dielectric constant (polarity) of the immediateenvironment in the binding pocket of the chlorophyll a molecule were defined for each cofactor bindingsite. In addition, the structural impact of the absence of one chlorophyll a molecule and the quality ofprotein folding were evaluated for each of these mutated polypeptides.
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