In vitro reconstitution of the photosystem I light-harvesting complex LHCI-730: heterodimerization is required for antenna pigment organization

Abstract

Here we describe the in vitro reconstitution of photosystem I light-harvesting complexes with pigments and proteins (Lhca1 and Lhca4) obtained by overexpression of tomato Lhca genes in Escherichia coli. Using Lhca1 and Lhca4 individually for reconstitution results in monomeric pigment-proteins, whereas a combination thereof yields a dimeric complex. Interactions of the apoproteins is highly specific, as reconstitution of either of the two constituent proteins in combination with a light-harvesting protein of photosystem II does not result in dimerization. The reconstituted Lhca1/4, but not complexes obtained with either Lhca1 or Lhca4 alone, closely resembles the native LHCI-730 dimer from tomato leaves with regard to spectroscopic properties, pigment composition, and stoichiometry. Monomeric complexes of Lhca1 or Lhca4 possess lower pigment/protein ratios, indicating that interactions of the two subunits not only facilitates pigment reorganization but also recruitment of additional pigments. In addition to higher averages of chlorophyll a/b ratios in monomeric complexes than in LHCI-730, comparative fluorescence and CD spectra demonstrate that heterodimerization involves preferential ligation of more chlorophyll b.

Figure 1

Polypeptide composition of inclusion bodies isolated from E. coli after overexpression of Lhca1 (lane 1), Lhca4 (lane 2), and AB96 (lane 3). The gel was stained with coomassie. MM, molecular mass standards.

Figure 2

Reconstitution analysis with individual E. coli-synthesized LHCI and LHCII apoproteins and combinations thereof. (A) Unstained gel obtained by nondenaturing gel electrophoresis. The presence (+) or absence (–) of the individual proteins in the reconstitution assay is depicted. (B) Fractionation pattern of the reconstitution mixtures in sucrose density gradients. The arrow points to the heterodimer band.

Figure 3

Polypeptide composition of the reconstituted Lhca complexes as well as of the native LHCI-730 and LHCII obtained by sucrose density gradients. Proteins were visualized by coomassie. MM, molecular mass standards.

Figure 4

77-K fluorescence spectra of native and reconstituted LHCI complexes. In A fluorescence emission spectra are presented that had been obtained by excitation at 440 nm for samples as indicated. In B excitation spectra are shown for fluorescence emissions at 687 nm (reconstituted Lhca1) or 734 nm (reconstituted Lhca4, reconstituted and native LHCI-730) after normalizing the 438-nm values. Samples with 10 μg Chl per ml were measured.

Figure 5

CD spectra of native and reconstituted LHCI pigment proteins. (A) Spectra for the native and reconstituted dimeric LHCI-730. (B) Corresponding spectra for monomeric complexes reconstituted from individual LHCI apoproteins. (C) The arithmetic sum of the two reconstituted monomeric complexes as well as the reconstituted dimeric complex. All spectra were obtained or calculated for samples with 50 μg Chl per ml.