Quantification of the forces that mediate electron transfers between proteins

介导蛋白质之间电子转移的力的量化

• 批准号: BB/P002005/1
• 负责人: Matthew Johnson
• 金额: $ 48.52万
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP002005%2F1
• 关键词: Quantification; forces; mediate; electron; transfers

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.

电子转移反应是光合作用和呼吸的基础，它为地球上的所有生命提供动力。本质上，太阳或食物直接提供的能量用于沿着蛋白质链移动电子。这些蛋白质中的一些可以自由移动，来回携带其电子货物往返于其他蛋白质的货物，这些蛋白质固定在一块薄膜上。这个谜团是一种自由移动的蛋白如何找到特定膜附着的蛋白质的方式，它如何在膜表面停靠，释放其电子，然后设法取消锁定，所有这些都以几毫秒为单位。然而，如果没有数百种这些电子转移反应每秒发生，地球上的生命就无法持续。这些蛋白质对以某种方式平衡了两个相互矛盾的要求：它们必须迅速而专门地集中到转移电子，但是它们也必须能够在之后迅速分离。因此，首先将蛋白质融合在一起的任何力都可以转换为反向 - 这是怎么可能的？这是什么开关？发现这一点是拟议研究的目的，它对地球上所​​有能源的电子传输具有重要意义。到目前为止，通过查看数十亿个蛋白质分子的集体行为，已经研究了蛋白质之间的电子转移反应。当电子之间移动时，这些蛋白质的光吸收特性会发生变化。这是因为这些蛋白质含有彩色的出血分子，例如血红蛋白中的血红蛋白。过去的工作，监测蛋白质的颜色及其电子货物的颜色，已经显示了整个分子种群的表现，但蛋白质就像人类一样是个体。每个分子都与其他分子略有不同。我们需要在单个蛋白质水平上了解这些生物反应，以便我们可以测量将它们融合在一起的力。问题是我们不知道单个蛋白质分子的行为，更重要的是，我们对将蛋白质融合在一起的有吸引力的力量以及在电子之间跳跃后将它们分开的驱虫力不了解。为了测量这些力，并发现允许对接/撤离的可逆开关，我们开发了一种将一种蛋白质伴侣（接收电子伴侣）连接到玻璃表面的方法。另一种蛋白质是一种带有电子的蛋白质，连接到探针的尖端上，该探针的尖端使其更靠近表面附着的蛋白质，直到电子在它们之间跳跃为止。该探针是一种称为原子力显微镜（AFM）的高度敏感仪器的一部分。当我们用电子接受蛋白从表面缩回AFM探针时，我们惊讶地发现我们遇到了一种电阻。为什么会发生这种情况？一旦电子转移，毫无疑问，尖端连接和表面连接的蛋白很容易拆开。看来我们跳了枪 - 拉得太早 - 我们等了足够长的时间，以至于蛋白质可以重组自己进行分离事件。因此，可逆开关允许对接，然后将电子传输，然后撤离尚未激活。现在，我们正处于可以使用AFM来了解单个蛋白质分子如何首先吸引彼此的位置，以及它们在电子传输后如何变化，以便它们可以解锁和分离。此外，我们可以使用只有在我们发光时才有效的电子蛋白质，以便我们可以准确控制这些反应何时发生。最后，我们可以制造具有改变接触区域的蛋白质，以找出蛋白质的哪些部分对于对接/撤离很重要。我们认为，这些测量值是其中的第一个测量值，它将告诉我们电子传输对植物光合作用和呼吸的必不可少，因此如此迅速有效地工作。

项目成果

Single-molecule study of redox control involved in establishing the spinach plastocyanin-cytochrome bf electron transfer complex

建立菠菜质体蓝素-细胞色素 bf 电子转移复合物的氧化还原控制的单分子研究

• DOI: 10.1016/j.bbabio.2019.06.013
• 发表时间: 2019
• 期刊: Biochimica et Biophysica Acta (BBA) - Bioenergetics
• 作者: Mayneord G