L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

21 août 19301930/08/21 (A31,N10841)

Structural models proposed for each trimer of Allophycocyanin.

<p>A) Schematic representation of the different composition trimers. B) Structure of APC, APC_1, APC_2, and APC_3.</p

Characterization of phycobilisomes of <i>Gracilaria chilensis</i>.

<p>A) Absorption(-) and emission(..) spectra. B) Transmission electron micrograph of purified phycobilisomes.The inserts show amplified images. Schematic drawings of PBS are also shown.</p

Spectroscopic characterization and oligomerization state of Allophycocyanin.

<p>A) Absorption (-) and emission (--) spectra of purified Allophycocyanin. B) Molecular sieve chromatogram; the standards and the sample are indicated.</p

Crystallographic structure of Allophycocyanin from <i>Gracilaria chilensis</i>.

<p>A) Ribbon representation of the asymmetric unit, the heterodimer. B) A section of the |2F-Fo| omit electron density map is shown for phycocyanobilin in α subunit. The residues interacting with the chromophore are also shown.</p

Multiple sequence alignment of γ³³ subunits available, with the phycoerythrin γ³³ subunits from of <i>G</i>. <i>ch</i>.

<p>In bold, the possible chromophorylation sites for conservation of cysteine residues demonstrated in <i>G</i>. <i>coulteri</i> (upper triangles) and <i>G</i>. <i>pacifica</i> (lower asterisk) are shown. 1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195656#pone.0195656.ref021" target="_blank">21</a>], 2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195656#pone.0195656.ref033" target="_blank">33</a>], 3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195656#pone.0195656.ref034" target="_blank">34</a>], 4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195656#pone.0195656.ref010" target="_blank">10</a>], 5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195656#pone.0195656.ref006" target="_blank">6</a>], 6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195656#pone.0195656.ref012" target="_blank">12</a>], 7 This work.</p

Absorption spectra of native and denatured R phycoerythrin subunits.

<p>A) α subunit, B) β subunit and C) γ³³ subunit. Dotted line: native sample; solid line: denatured sample.</p

The γ³³ subunit of R-phycoerythrin from <i>Gracilaria chilensis</i> has a typical double linked phycourobilin similar to γ subunit

<div><p>Phycobilisomes (PBS) are accessory light harvesting protein complexes formed mainly by phycobiliproteins (PBPs). The PBPs absorb light that is efficiently transferred to Photosystems due to chromophores covalently bound to specific cysteine residues. Besides phycobiliproteins (PE), the PBS contains linker proteins responsible for assembly and stabilization of the whole complex and the tuning of energy transfer steps between chromophores. The linker (γ³³) from <i>Gracilaria chilensis</i>, is a chromophorylated rod linker associated to (αβ)₆ hexamers of R-phycoerythrin (R-PE). Its role in the energy transfer process is not clear yet. Structural studies as well as the composition and location of the chromophores are essential to understand their involvement in the energy transfer process in PBS. To achieve this, the coding gene of γ³³ was cloned and sequenced. The sequence was analyzed by informatics tools, to obtain preliminary information which leaded the next experiments. The protein was purified from R-phycoerythrin, and the sequence confirmed by mass spectrometry. The coding sequence analysis revealed a protein of 318 aminoacid residues containing a chloroplastidial transit peptide (cTP) of 39 aminoacids at the N-terminus. The conservation of cysteines revealed possible chromophorylation sites. Using α and β R-PE subunits as spectroscopic probes in denaturation assays, we deduced a double bonded phycourobilin (PUB) on γ³³ subunit that were confirmed between Cys62 and Cys73 (DL-PUB^62/73) by mass spectrometry. The cysteines involved in the double link are located in a helical region, in a conformation that reminds the position of the DL-PUB^50/61 in the β subunit of R-PE. The position of single linked PUB at Cys⁹⁵ and a single linked PEB at Cys¹⁷² were also confirmed. Spectroscopic studies show the presence of both types of chromophores and that there are not energy transfer by FRET among them.</p></div

Detail of B-helix on β-PE subunit from crystallographic model PDB-1EYX.

<p>The sequences involving on β-DL-PUB^50/61 and γ³³-DL-PUB^62/73 are shown for comparison.</p

Sequence comparison between α and α^II (ApcA and ApcD), β and β¹⁸ (Apcb and ApcF) and α and the PB_domain of the L_CM (ApcA and ApcE).

<p>Chromophore binding region are enclosed in green rectangles, and the conserved residues are highlighted in green. The cysteine residues that bind the chromophores are shown in red background. The PB-loop sequence is enclosed in blue lines.</p