Laboratorio de Biofísica Molecular, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas Universidad de Concepción, Concepción, Chile.
Michael Swann Building, Kings' Buildings, University of Edinburgh, Edinburgh, Scotland, United Kingdom.
PLoS One. 2018 Apr 10;13(4):e0195656. doi: 10.1371/journal.pone.0195656. eCollection 2018.
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-PUB62/73) 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-PUB50/61 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.
藻胆体（PBS）是主要由藻胆蛋白（PBPs）组成的辅助光收集蛋白复合物。PBPs 吸收的光能由于与特定半胱氨酸残基共价结合的发色团而有效地转移到光系统中。除了藻胆蛋白（PE）外，PBS 还包含连接蛋白，负责组装和稳定整个复合物，并调整发色团之间的能量转移步骤。来自智利石花菜的连接蛋白（γ33）是一种与藻红蛋白（R-PE）的（αβ）6 六聚体相关的发色团化棒状连接蛋白。它在能量转移过程中的作用尚不清楚。结构研究以及发色团的组成和位置对于理解它们在 PBS 中的能量转移过程至关重要。为了实现这一目标，克隆并测序了 γ33 的编码基因。通过信息学工具对序列进行分析，获得了初步信息，为下一步实验提供了指导。从 R-藻红蛋白中纯化了该蛋白，并通过质谱法确认了序列。编码序列分析表明，该蛋白由 318 个氨基酸残基组成，在 N 端含有一个 39 个氨基酸的叶绿体转运肽（cTP）。保守半胱氨酸揭示了可能的发色团化位点。在变性实验中使用 α 和 β R-PE 亚基作为光谱探针，我们推断出 γ33 亚基上存在一个双键藻红胆素（PUB），通过质谱法证实其位于 Cys62 和 Cys73 之间（DL-PUB62/73）。参与双键的半胱氨酸位于一个螺旋区域，其构象类似于 R-PE β 亚基中 DL-PUB50/61 的位置。还证实了 Cys95 处的单键 PUB 和 Cys172 处的单键 PEB 的存在。光谱研究表明存在这两种类型的发色团，并且它们之间没有通过 FRET 进行能量转移。
