Quantification of the forces that mediate electron transfers between proteins

电子转移反应是光合作用和呼吸作用的基础，而光合作用和呼吸作用是地球上所有生命的动力。本质上，由太阳或食物直接提供的能量被用来沿着蛋白质链移动电子；其中一些蛋白质可以自由移动，来回穿梭携带电子往返于其他蛋白质，这些蛋白质被固定在一层薄膜中。令人费解的是，自由移动的蛋白质是如何找到特定的附着在膜上的蛋白质的，它如何停靠在膜表面，释放电子，然后设法脱离停靠，所有这一切都在几毫秒内完成。然而，如果没有每秒数百次这样的电子转移反应，地球上的生命就不可能持续存在。不知何故，这些蛋白质对平衡了两个相互冲突的要求：它们必须迅速聚集在一起，特别是为了转移电子，但它们也必须能够在之后迅速分离。因此，无论是什么力量将蛋白质聚集在一起，都可以转换为反向--这是怎么可能的？这个开关是什么？找出这一点是这项拟议研究的目的，它对地球上所有产生能量的电子转移都有重要影响。到目前为止，蛋白质之间的电子转移反应是通过观察数十亿个蛋白质分子的集体行为来研究的。当电子在它们之间移动时，这些蛋白质的光吸收特性就会改变；这是因为这些蛋白质含有有色的血红素分子，就像血液中的血红蛋白一样。过去的工作，监测蛋白质的颜色，从而监测它们的电子运输量，已经显示了整个分子群体的行为，但蛋白质是个体，就像人类一样；每个分子都略有不同。我们需要在单个蛋白质的水平上理解这些生物反应，这样我们才能测量将它们聚集在一起的力量。问题是，我们不知道单个蛋白质分子的行为，更重要的是，我们对将蛋白质聚集在一起的吸引力和电子在它们之间跳跃后分离它们的排斥力一无所知。为了测量这些力，并发现允许对接/脱离对接的可逆开关，我们开发了一种方法，将一个接收电子的蛋白质伙伴连接到玻璃表面。另一种携带电子的蛋白质被连接到一个探针的尖端，该探针被拉近与表面附着的蛋白质越来越近，直到电子在它们之间跳跃。该探头是一种名为原子力显微镜(AFM)的高灵敏度仪器的一部分。当我们将原子力显微镜探针与接受电子的蛋白质一起从表面收回时，我们惊讶地发现我们遇到了阻力。为什么会发生这种事？当然，一旦电子转移，末端附着和表面附着的蛋白质将很容易被撕裂。看起来我们似乎操之过急了--拔得太早--我们没有等足够长的时间来等待蛋白质为分离事件进行自我重组。因此，允许对接，然后电子转移，然后脱离对接的可逆开关还没有激活。我们现在可以使用原子力显微镜来找出单个蛋白质分子最初是如何相互吸引的，以及它们在电子转移后如何变化，以便它们可以脱离和分离。此外，我们可以使用一种接受电子的蛋白质，它只有在我们照射到它的时候才起作用，这样我们就可以准确地控制这些反应发生的时间。最后，我们可以制造具有改变接触区的蛋白质，以找出蛋白质的哪些部分对对接/脱离对接是重要的。我们认为，这些测量将告诉我们，对植物光合作用和呼吸至关重要的电子转移是如何如此迅速和有效地发挥作用的，这是此类测量中的第一次。

Electron transfer reactions are the basis of photosynthesis and respiration, which power all life on Earth. In essence energy directly provided by the sun or from foodstuffs is used to move electrons along a chain of proteins; some of these proteins can move freely, shuttling back and forth carrying their cargo of electrons to and from other proteins that are held in position within a thin sheet of membrane. The mystery is how a freely-moving protein finds its way to a particular membrane-attached protein, how it docks at the membrane surface, releases its electron and then manages to undock, all in a few milliseconds. Yet without hundreds of these electron transfer reactions happening every second, life on Earth could not be sustained. Somehow these pairs of proteins balance two conflicting requirements: they have to come together quickly and specifically to transfer electrons, yet they also have to be able to separate rapidly afterwards. So whatever forces brought the proteins together in the first place can be switched into reverse - how is this possible? What is this switch? Finding this out is the purpose of the proposed research, and it has important implications for all energy-yielding electron transfers on Earth. Up until now, electron transfer reactions between proteins have been studied by looking at the collective behaviour of billions of protein molecules. The light-absorbing properties of these proteins changes when electrons move between them; this is because these proteins contain a coloured haem molecule, as in haemoglobin in blood. Past work, monitoring the colour of the proteins and therefore their cargo of electrons, has shown how whole populations of molecules behave, but proteins are individuals just like humans; every molecule is slightly different from the others. We need to understand these biological reactions at the level of individual proteins so we can measure the forces that bring them together. The problem is that we don't know how individual protein molecules behave, and more importantly we don't know anything about the attractive forces that bring the proteins together and the repelling forces that separate them after the electron has jumped between them. To measure these forces, and to discover the reversible switch that allows docking/undocking, we developed a method to attach one protein partner, the one that receives the electrons, to a glass surface. The other protein, the one carrying the electron, was attached to the tip of a probe that was brought closer and closer to the surface-attached protein until the electron jumps between them. This probe is part of a highly sensitive instrument called an atomic force microscope (AFM). When we retracted the AFM probe from the surface with the electron accepting proteins we were surprised to find that we met a resistance. Why would this happen? Surely the tip-attached and surface-attached proteins would be easy to pull apart once the electron has transferred. It looks as if we had jumped the gun - pulled too early - and we had not waited long enough for the proteins to reorganise themselves for the separation event. So the reversible switch that allows docking, then electron transfer, then undocking had not been activated yet. We are now in the position where we can use our AFM to find out how single protein molecules attract each other in the first place and how they change after electron transfer in order that they can undock and separate. Moreover we can use an electron-accepting protein that only works when we shine light on it so we can control exactly when these reactions occur. Finally, we can make proteins with altered contact zones to find out which parts of the protein are important for docking/undocking. We think that these measurements, the first of their kind, will tell us how electron transfers, essential for plant photosynthesis and for our respiration, work so quickly and efficiently.