Fig 1.
(A) Chromatographic separation of the subunits components of R-phycoerythrin complexes from G. ch. by RP-HPLC (B) Electrophoretic assay of the purified subunits, in 10% SDS-Tricine Gel, stained with Coomassie blue (right) and exposed to UV light to determine presence of chromophores (left).
Fig 2.
In silico translation of phycoerythrin γ33 subunit from G. ch.
MS corresponds to the sequence confirmed by mass spectrometry. The chloroplast transport peptide at N-terminal is shown in cursive letters. The internal repeats are highlighted in bold letters. The arrowhead represents the start of the sequence of the mature protein.
Fig 3.
Absorption and emission spectra of the purified γ33.
Continuous line shows the absorption spectrum. Dashed and dotted lines show the emission spectra of γ33 subunit after excitation at 490nm and 538nm respectively. Insert emission spectrum of R-Phycoerythrin after excitation at 490 and 540 nm.
Fig 4.
Multiple sequence alignment of γ33 subunits available, with the phycoerythrin γ33 subunits from of G. ch.
In bold, the possible chromophorylation sites for conservation of cysteine residues demonstrated in G. coulteri (upper triangles) and G. pacifica (lower asterisk) are shown. 1 [21], 2 [33], 3 [34], 4 [10], 5 [6], 6 [12], 7 This work.
Fig 5.
Absorption spectra of native and denatured R phycoerythrin subunits.
A) α subunit, B) β subunit and C) γ33 subunit. Dotted line: native sample; solid line: denatured sample.
Fig 6.
MALDI-TOF mass spectra of PUB-peptide.
A) Peptide isolated from tryptic digestion and cyanogen bromide cleavage of native γ33 phycoerythrin. The insert shows absorption spectrum of the PUB-peptide. B) Structural scheme of PUB-peptide identified by MS-Bridge of the double linked PUB to cysteines on γ33.
Fig 7.
Detail of B-helix on β-PE subunit from crystallographic model PDB-1EYX.
The sequences involving on β-DL-PUB50/61 and γ33-DL-PUB62/73 are shown for comparison.