Elucidating the transient nature of electron transfer complexes at the single-molecule level

电子转移反应是光合作用和呼吸作用的基础，为地球上的所有生命提供能量来源。由太阳或食物直接提供的能量被用来使电子沿着蛋白质链移动以释放能量。参与电子传递链的一些蛋白质可以自由地来回移动，携带电子往返于固定在生物膜薄片内的伴侣蛋白质。自由移动的电子载体蛋白必须与其适当的膜附着伴侣快速和特异地配对，以确保有效的电子转移，同时该对必须能够在电子转移过程完成后足够迅速地分离，以便该过程每秒可以重复数百次。我们的研究将有助于回答有关的力量，直接和带来的伙伴蛋白质;如何做的电子载体停靠在膜表面，以及它们是如何在几微秒后，电子转移发生释放的问题。当蛋白质必须分离时，是什么开关逆转了它们之间的相互作用？传统上，蛋白质之间的电子转移反应已经通过观察大集合的光学性质来研究。这些蛋白质含有一种有色血红素分子，类似于血液中的血红蛋白，当电子在它们之间移动时，分子的光吸收特性就会发生变化。通过监测蛋白质的颜色，以及它们的电子货物，我们可以看到这些蛋白质是如何集体行为的。在我们提出的工作中，我们的目标是进一步研究单个蛋白质水平上的电子转移反应。我们在将这些蛋白质聚集在一起的吸引力和在电子在它们之间跳跃后将它们分开的排斥力方面的知识有很大的差距。我们不知道蛋白质或周围环境的性质如何影响分子之间的相互作用，以及这些因素如何影响电子转移过程的效率。为了测量蛋白质之间的相互作用力和实际电子转移，我们开发了一种方法，人工将两个电子转移伙伴放在一起。接收电子的蛋白质（受体）附着在玻璃表面，而携带电子的蛋白质（供体）附着在探针的非常尖锐的尖端，可以在空间中非常精确地定位。将探针带到表面附着的蛋白质上可以使电子从供体跳到受体。这种探针是一种称为原子力显微镜（AFM）的高灵敏度仪器的一部分，它也可以测量探针和衬底之间的电流。当我们将AFM探针从表面缩回时，我们可以测量抵抗两种蛋白质分离的力。与此同时，我们可以通过测量探针和表面之间的电流来监测电子在蛋白质之间跳跃的容易程度。通过这种实验方法，我们可以使用我们的AFM来发现单个蛋白质分子最初是如何相互吸引的，以及电子转移后它们的相互作用如何变化，以便它们能够脱离和分离。此外，我们可以使用遗传修饰的电子接受蛋白质，看看两种蛋白质接触的位点的特定变化如何影响蛋白质对接在一起的可能性，并找出这些变化如何影响电子转移反应的效率以及受体和供体蛋白质伴侣的后续解偶联。

Electron transfer reactions are the basis of photosynthesis and respiration, which provide the energy source for all life on Earth. The energy directly provided by the sun or from foodstuffs is used to move electrons along a chain of proteins in order to release energy. Some of the proteins involved in the electron transfer chain can move freely back and forth carrying electrons to and from their partner proteins fixed within a thin sheet of biological membrane. The freely-moving electron carrier proteins have to pair with their appropriate membrane-attached partners quickly and specifically to ensure efficient electron transfer, while at the same time the pair has to be able to separate rapidly enough after the electron transfer process is complete so that the process can be repeated hundreds of times each second. Our investigation will help to answer questions concerning the forces that direct and bring together the partner proteins; how do the electron carriers dock at the membrane surface and how they are released in a few microseconds after the electron transfer takes place? What is the switch that reverses the interactions between the proteins when they have to separate? Traditionally, electron transfer reactions between proteins have been studied by looking at the optical properties of large ensembles. These proteins contain a coloured haem molecule, similar to haemoglobin in the blood, and the light-absorbing properties of the molecules change when electrons move between them. Monitoring the colour of the proteins, and therefore their cargo of electrons has shown how these proteins behave collectively. In the work we are proposing, we aim to take a step further and to study the electron transfer reactions at the level of individual proteins. We have a significant gap in our knowledge regarding the attractive forces that bring these proteins together and the repelling forces that separate them after the electron has jumped between them. We do not know how the properties of the proteins or the surrounding environment affect the interactions between the molecules and how these factors affect the efficiency of the electron transfer process.To measure the interaction forces and the actual electron transfer between the proteins, we developed a method to artificially bring the two electron transfer partners together. The protein that receives the electrons (the acceptor) is attached to a glass surface, while the protein that carries the electron(the donor) is attached to a very sharp tip of a probe that can be positioned very precisely in space. Bringing the probe to the surface-attached protein allows the electron to jump from the donor to the acceptor. This probe is part of a highly sensitive instrument called an atomic force microscope (AFM), which can also measure the current passing between the probe and the substrate. When we retract the AFM probe from the surface we can measure the forces that resist the separation of the two proteins. At the same time, we can monitor how easy it is for the electron to jump between the proteins, by measuring the current between the probe and the surface.With this experimental approach, we can use our AFM to find out how single protein molecules attract each other in the first place and how their interaction changes after electron transfer so that they can undock and separate. Moreover, we can use genetically-modified electron-accepting proteins and see how particular changes in the sites where the two proteins come into contact would affect the likelihood of the proteins docking together, and to find out how these changes would affect the efficiency of the electron transfer reactions and the subsequent uncoupling of the acceptor and donor protein partners.