Photosystem Two accessory proteins: structures binding sites and functions

光系统二（PSII）复合体被正确地描述为“生命引擎”。在植物、藻类和蓝藻中发现的这种分子机器能够利用阳光的能量将非常稳定和丰富的水分子分解成分子氧，分子氧作为副产品释放到大气中，质子用于产生ATP，“高能”电子最终与ATP一起用于将大气中的二氧化碳固定为糖分子。这些碳水化合物可以用作燃料，并合成生物质用于生长。多年来，为了解PSII的结构及其工作原理，人们付出了相当大的努力。现在已知PSII由超过20种单独的蛋白质组成，它们在脂质膜中结合在一起成为一个大的蛋白质复合物。与这种复合物紧密相关的是收集太阳能的色素，可以传输高能电子的小有机分子和包含1个钙离子和4个锰离子的金属簇。正是在这个深埋在蛋白质复合物中的金属簇上，水结合并氧化成分子氧，放弃其电子。但PSII并不是一台完美的机器;它有时会出现故障，特别是在阳光非常明亮的时候，必须进行修理。为了做到这一点，受损的PSII复合物被部分分解成一个较小的复合物，受损的蛋白质被识别，被膜内发现的特殊蛋白酶特异性降解，插入一个新的蛋白质，复合物重新组装。如果没有这种特殊的修复机制，PSII将在光照下迅速失活，植物的生长和放氧将受到抑制。我们研究的目的是了解PSII的功能，PSII是如何从其组成部分组装，以及如何有效地修复。了解这些过程可能使我们在未来能够增强作物的光合作用，以便我们能够增加生长，以帮助满足对更多食物和更多生物质日益增长的需求。这一知识也可能应用于设计新的可持续除草剂或设计新的人造催化剂，这些催化剂可能作为“人造叶子”，从太阳能中提供可再生燃料。以前的工作已经确定了一些小的蛋白质，似乎参与组装，修复或PSII的最佳功能。我们建议确定这些“辅助”蛋白的结构，这样我们就可以看到它们在空间中是如何折叠的，以及它们如何与PSII结合。为了帮助做到这一点，我们在细菌中制造了大量的目标蛋白质，纯化了蛋白质并制造了晶体，我们可以在X射线衍射实验中使用晶体来确定结构。或者，我们也可以使用核磁共振（NMR）光谱，其优点是不需要制造晶体。我们也已经能够使一些辅助蛋白质和完整的氧释放PSII复合物或更小的蛋白质片段之间的复合物。通过X射线晶体学或NMR分析这些复合物将使我们能够第一次观察这些辅助蛋白如何在分子水平上与PSII结合。将这些结构相互作用与细胞中发生的事情联系起来是很重要的。为了做到这一点，我们制造了一种缺乏这些辅助蛋白的蓝细菌菌株。通过研究这些菌株的PSII组装和修复以及PSII活性，我们希望能够确定PSII中与结构结果相关的精确缺陷。通过这种方式，我们将能够了解有关“生命引擎”如何组装和保持工作状态的宝贵新信息。

The Photosystem Two (PSII) complex has quite rightly been described as the 'Engine of Life'. This molecular machine which is found in plants, algae and cyanobacteria is able to use the energy of sunlight to split very stable and abundant water molecules into molecular oxygen, which is released into the atmosphere as a by-product, protons which are used to generate ATP, and 'high energy' electrons which are ultimately used with the ATP to fix atmospheric carbon dioxide into sugar molecules. These carbohydrates can then be used as a fuel and to synthesise biomass for growth. Over the years there has been considerable effort to understand the structure of PSII and how it works. It is now known that PSII is made up of over 20 individual proteins, bound together as a large protein complex in a lipid membrane. Tightly associated with this complex are pigments that harvest the solar energy, small organic molecules that can transport high-energy electrons and a metal cluster comprising one calcium and 4 manganese ions. It is at this metal cluster, buried deep in the protein complex, that water binds and is oxidised to molecular oxygen, giving up its electrons. But PSII is not a perfect machine; it sometimes breaks down, especially when the sunlight is very bright, and has to be repaired. To do this the damaged PSII complex is partially disassembled into a smaller complex, and the damaged protein is recognised, specifically degraded by special proteases found within the membrane, a new protein inserted and the complex reassembled. Without this special repair mechanism PSII would be quickly inactivated in the light and plant growth and oxygen evolution would be inhibited. The purpose of our research is to understand how PSII functions, how PSII is assembled from its component parts and how it is repaired efficiently. Understanding these processes might allow us in the future to enhance photosynthesis in crop plants so that we can increase growth to help satisfy the ever increasing demand for more food and more biomass. This knowledge might also have applications in the design of new, sustainable herbicides or the design of new man-made catalysts that might act as 'artificial leaves' to provide renewable fuels from solar energy. Previous work has identified a number of small proteins that seem to be involved in the assembly, repair or optimal functioning of PSII. We propose to determine the structures of these 'accessory' proteins, so we can see how they are folded in space, and how they bind with PSII. To help do this we have made large amounts of our target proteins in a bacterium, purified the proteins and made crystals which we can use in X-ray diffraction experiments to determine the structure. Alternatively we can also use nuclear magnetic resonance (NMR) spectroscopy which has the advantage that crystals need not be made. We have also been able to make complexes between some of the accessory proteins and either the intact oxygen-evolving PSII complex or smaller protein segments. Analysis of these complexes by x-ray crystallography or NMR will allow us, for the first time, to observe how these accessory proteins engage with PSII at the molecular level. It is important to relate these structural interactions with what is happening in the cell. To do this we have made strains of a cyanobacterium that lack these accessory proteins. By studying PSII assembly and repair and PSII activity in these strains we hope to be able to identify a precise defect in PSII that can be related to the structural results. In this way we will be able learn valuable new information on how the 'Engine of Life' is assembled and maintained in a working state.