Fig 1.
In silico analysis of LHCBM proteins.
Multiple sequence alignment of C. reinhardtii LHCBM proteins (Clustal OMEGA). Red-colored amino acids indicate the conserved Chl a and Chl b binding sites and the neoxanthin binding site (tyrosine, N1) [36]; green colored amino acids indicate the transit peptide; blue colored amino acids indicate the region of the trimerization motif.
Table 1.
Isoeletric point and number of sulfur atoms of the LHCBM proteins.
Fig 2.
Tricine SDS-PAGE of the ndIEF fractions.
(A) Monomeric LHCII and (B) Trimeric LHCII separated by ndIEF. (C) Monomeric LHCII and (D) Trimeric LHCII ndIEF fractions separated by SDS-PAGE. MM: Molecular Marker; Mon LHCII: Monomeric LHCII isolated from a sucrose gradient purification. B2FI, B2FII, B2FIII: Different fractions from the LHCII monomer. B3FI, B3FII, B3III: Different fractions from the LHCII trimers.
Fig 3.
Spectroscopic analysis of the ndIEF fractions.
(A) and (B): Absorption spectra of ndIEF fractions. (C) and (D): Fluorescence emission spectra at 77K (excitation at 440 nm). (E) and (F): Circular dichroism spectra at 10°C. B2FI, B2FII, B2FIII: Different fractions from the LHCII monomer. B3FI, B3FII, B3III: Different fractions from the LHCII trimers.
Table 2.
Pigment composition of the LHCII fractions generated by ndIEF.
Fig 4.
Time-Resolved Fluorescence Decay kinetics of the ndIEF fractions.
(A) monomeric complexes; (B) trimeric complexes.
Table 3.
Excited state Lifetimes of the LHCBM monomeric and trimeric fractions.
Fig 5.
Absorption spectra (A)–(B), Fluorescence emission spectra at 77K (excitation at 440 nm) (C) and Circular Dichroism (D) of the LHCBM reconstituted complexes.
The absorptions are normalized to the Qy peaks. Fluorescence emission spectra are normalized to the maximum, while the CD signals are normalized on the absorption spectra.
Table 4.
Pigment composition of the LHCBM reconstituted proteins in vitro.
Fig 6.
Changes in the CD signal of the LHCBM reconstituted proteins at 680 nm as a function of temperature.
Black line represents the fitting of the denaturation.
Table 5.
Decay kinetics of the different LHCBM reconstituted proteins.
Fig 7.
Comparison of the absorption (A–C) and Circular Dichroism (B–D)) of LHCBM1, LHCBM2 reconstituted and the purified fractions from ndIEF containing LHCBM1 and LHCBM2/7.
The absorptions are normalized to the Qy peaks, while the CD signals are normalized on the absorption spectra.
Table 6.
Decay kinetics of LHCBM1 reconstituted in vitro at different pH.