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

by Aleikar Vásquez-Suárez, Francisco Lobos-González, Andrew Cronshaw, José Sepúlveda-Ugarte, Maximiliano Figueroa, Jorge Dagnino-Leone, Marta Bunster, José Martínez-Oyanedel

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 Gracilaria chilensis, 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.

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