蓝藻藻胆体的冷冻电镜结构生物学研究

Phycobilisomes (PBSs) attach to cytoplasmic side of thylakoid of cyanobacterial and red algae cell. PBSs structurally interact with PSII and PSI within thylakoid with their bottoms, functionally harvest and transfer energy to two photosynthetic reaction centers with high efficiency. PBSs are composed of phycobiliprotein and linker proteins. Linkers are structurally necessary for self-assembling of intact phycobilisome, and functionally influence their adjacent bilins and energy transfer pathway. PBSs in Red Algae are mostly block-shaped and hemi-ellipsoidal, while PBSs in most of cyanobacteria are most hemidiscoidal and bundle-shaped. Linkers in Red Algae and cyanobacteria are structurally different, which also results in different PBSs assembling and energy transferring mechanisms. Gloeobacter violaceus own important position in evolution of cyanobacteria, which determines its structural differences of LRC and special PBSs assembling mechanism. Red Algal PBSs have been resolved at near-atomic resolutions by cryo-EM technique, but cyanobacterial PBSs have not been resolved. Because their flat shapes result in great preferential orientation problems during cryo-sampling, which bring great difficulties for their structures to be resolved by cryo-EM. To solve the high-resolution structure of whole cyanobacterial PBSs is the key to understand the differences of structural mechanism, energy transferring mechanism within PBSs evolutionally from cyanobacteria to Red Algae.

蓝藻和红藻细胞的捕光复合物藻胆体结合在类囊体膜外基质侧，结构上以其底面与类囊体膜内PSII和PSI等相互作用，功能上捕获光能并高效传递给2个光反应中心。藻胆体由藻胆蛋白和连接蛋白构成。连接蛋白在结构上对藻胆体自组装机制不可或缺，功能上影响藻胆色素构象与能量传递途径。红藻藻藻胆体多为块状和半椭球状，蓝藻藻胆体则大部分为半圆盘状和束状；红藻和蓝藻连接蛋白结构差异，也导致藻胆体结构组装和能量传递的差异。Gloeobacter violaceus在蓝藻中重要的进化地位，决定了其核杆连接蛋白LRC结构差异，及其藻胆体组装形式的特殊性。红藻藻胆体近原子分辨率结构已被冷冻电镜技术解析，但蓝藻藻胆体由于其特殊的扁平结构，冷冻制样时存在严重的优势取向问题，导致其较难通过冷冻电镜解析结构。蓝藻完整藻胆体结构的高分辨结构解析，是进化层面上理解蓝藻-红藻藻胆体结构机制、及能量传递功能机制差异的关键。

本项目旨在通过单颗粒冷冻电镜技术，结合分子、生化、光谱等生物物理学方法研究最原始蓝藻G. violaceus藻胆体的结构以及能量传递机制。我们按原计划利用冷冻电镜单颗粒技术解析不同光照条件下的G. violaceus的束状藻胆体的短杆状态、长杆状态、最长杆状态的3个结构形式，分辨率分别为2.80 Å、2.96 Å、3.37 Å，并在此基础上搭建了高可信度的原子模型。其中短杆状态藻胆体包含8种连接蛋白，7种藻胆蛋白，一共由334个亚基构成。LCM辅助五个圆柱构成的核与高等蓝藻略有差异，杆内部LRCpcC2独特的N-Pfam01383-Pfam00427-C结构域排序导致了不同于高等蓝藻的独特长杆组装方式。首次得到了束状藻胆体特有的2种核杆连接蛋白LRC91的全长结构和LRC81的部分结构，它们在束状藻胆体结构组装中发挥重要作用。并且进一步分析了短杆的444个、长杆的876个色素的空间排布和部分独特的能量传递途径。结合已有全部藻胆体结构和系统发育树分析，未来可推测藻胆体结构进化机制脉络图。在本项目的资助下，我们还对紫球藻的红藻细胞，通过冷冻聚焦离子束减薄技术（cryo-FIB milling）联合冷冻断层三维重构技术（in situ cryo-ET技术）首次解析得到天然状态下的红藻P. purpureum细胞内原位的藻胆体-光系统II复合体13.2 Å分辨率的结构，为藻胆体捕获的光能传递给光系统的机制研究提供了结构生物学基础。