单颗粒冷冻电子显微学研究完整藻胆体的高分辨率结构

The initial process of photosynthesis is to harvest and transfer solar energy to photosynthetic reaction centers with high efficiency. Marine red algae, whose habitats are dim light under water, efficiently harvest light by their light harvesting antenna phycobilisome. Phycobilisomes, as one of two main kinds of light harvesting complexes of oxygen photosynthesis, structurally being composed of phycobiliproteins and linker proteins. The three types of phycobiliproteins covalently bind different numbers and types of bilins, but have similar structure of protein scaffold. Phycobiliproteins self-assemble in highly order to an intact phycobilisome whose structure stability relies on the participation of linker proteins. The solar transfer efficiency between phycobilisomes and PSII reaction centers could reach nearly 100%. Researches on phycobilisome structures have been going on for more than half a century, but none of their intact structure within near-atom resolution is achieved. Our research focuses on phycobilisome in Griffithsia pacifica, one representative species of marine red algae. To obtain phycobilisome structure with high-resolution, firstly, we adopted the recently developed Cryo– Electron Microscopy (Cryo– EM) technique, and followed workflow of cryogenic sample preparation optimization and 3-D reconstruction. Then, we sequence its transcriptome, and ensure the sequences by molecular biology technique. We expect to build a more confident 3-D mode within atom resolution. Structure of intact phycobilisomes not only structurally solve problem of how phycobilisomes self-assemble which confused scientists for half a century, but also provides solid evidence of how these gigantic protein-pigment complexes harvest light efficiently.

光合作用的第一步是高效地捕获和传递光能，水下弱光环境生长的海洋红藻依赖于藻胆体高效捕光。作为有氧光合作用捕光复合物两大类型之一，其结构上主要由藻胆蛋白和连接蛋白构成。结合不同数目种类的色素、且结构类似的三种藻胆蛋白以不同寡聚形式，加上各种连接蛋白，自发组装为高度有序完整藻胆体结构，功能上以近100%效率捕获和传递光能至光合反应中心。藻胆体结构研究开展很早，但至今仍未有完整藻胆体近原子分辨率的结构报道。本项目以太平洋凋毛藻为代表的海洋红藻的藻胆体为研究对象，采用最新发展的单颗粒冷冻电子显微学技术，通过冷冻制样和三维重构一系列流程获得高分辨率的解析；再对该藻转录组测序和分子生物学验证，搭建高可信度的原子模型。本研究不仅解决藻胆体结构组装难题，也为完善捕光色素蛋白的能量传递机制提供了重要的科学依据。

蓝藻和红藻的捕光复合物藻胆体结合在类囊体膜基质侧，与类囊体膜内PS II和PSI等相互作用，捕获光能并以大于98 %的效率传递给两个光反应中心。藻胆体由藻胆蛋白和连接蛋白构成。在本项目的支持下，我们采用冷冻透射电子显微镜（Cryo-EM）技术获得红藻G. pacific块状藻胆体3.5 Å分辨率的电子密度图，结合基因测序、质谱等方法得到蛋白序列并搭建了模型，首次得到全部20种全长连接蛋白结构。该复合体理论分子量为18.0MDa，包含862个蛋白亚基（25种蛋白），2048个色素分子。该复合体结构的解析揭示了完整藻胆体的组装方式，但受限于分辨率，色素分子的调控方式还是未知。为了解决这个问题，我们进一步解析了紫球藻P. purpureum 半椭球形藻胆体的结构，分辨率达到2.82Å。基于该高分辨率结构，我们仔细分析了色素分子和周围蛋白之间的相互作用，发现连接蛋白广泛地参与了色素分子能量状态的调节，即一些色素分子会与周围连接蛋白上的芳香族氨基酸相互作用，从而改变色素分子的能量状态，以确保能量的有效单向传递。这些研究成果为阐明藻胆体独特的光能捕获、传递和转化机制提供了重要基础，同时为人工模拟光合作用研究提供了新理论依据。