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

阐明单分子水平上电子转移复合物的瞬态性质

• 批准号: BB/V006630/1
• 负责人: Matthew Johnson
• 金额: $ 58.67万
• 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: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV006630%2F1
• 关键词: Elucidating; transient; nature; electron; transfer

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.

电子转移反应是光合作用和呼吸的基础，它为地球上所有生命提供了能量来源。太阳或食物直接提供的能量用于沿蛋白质链移动电子，以释放能量。电子传输链中涉及的一些蛋白质可以来回移动电子，从而从固定在薄片生物膜上的伴侣蛋白上移动。自由移动的电子载体蛋白必须与合适的膜附着的合作伙伴配对，以确保有效的电子传递，同时，两人必须能够在电子传输过程完成后能够足够快地分离，以便可以每秒重复数百次。我们的调查将有助于回答有关指导和汇集伴侣蛋白质的力量的问题；电子载体如何停靠在膜表面，以及在电子传输发生后如何在几微秒内释放它们？当蛋白质必须分开时，什么是逆转蛋白质之间的相互作用的开关？传统上，通过查看大型合奏的光学特性研究了蛋白质之间的电子转移反应。这些蛋白质含有有色的出血分子，类似于血液中的血红蛋白，当电子之间移动时，分子的光吸收特性会发生变化。监测蛋白质的颜色，因此它们的电子货物已经显示了这些蛋白质的行为方式。在我们提出的工作中，我们的目标是进一步迈出一步，并研究单个蛋白质水平的电子转移反应。关于将这些蛋白质融合在一起的吸引力的吸引力，我们的知识有很大的差距，在电子之间跳跃之后将它们分开的排斥力。我们不知道蛋白质或周围环境的特性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传递过程的效率。为了测量相互作用力和蛋白质之间的实际电子转移，我们开发了一种人为地将两个电子转移伙伴一起将两个电子转移的方法。接收电子（受体）的蛋白质连接到玻璃表面，而带有电子（供体）的蛋白质连接到探针的非常尖锐的尖端，该探针可以非常精确地放在太空中。将探针带到表面附着的蛋白质上，使电子从供体跳到受体。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，该仪器还可以测量探针和底物之间的电流。当我们从表面缩回AFM探针时，我们可以测量抵抗两种蛋白质分离的力。同时，我们可以通过测量探针和表面之间的电流来监视电子在蛋白质之间跳跃之间的容易。采用这种实验方法，我们可以使用AFM来找出单个蛋白质分子首先在彼此中互相吸引，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，我们可以使用遗传改性的电子感知的蛋白质，并查看两种蛋白质接触的位点中如何变化会影响蛋白质对接在一起的可能性，并了解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蛋白蛋白parters的后续脱离。

项目成果

Cryo-EM structures of the Synechocystis sp. PCC 6803 cytochrome b6f complex with and without the regulatory PetP subunit.

• DOI: 10.1042/bcj20220124
• 发表时间: 2022-07-15
• 期刊: The Biochemical journal

Changes in supramolecular organization of cyanobacterial thylakoid membrane complexes in response to far-red light photoacclimation.

• DOI: 10.1126/sciadv.abj4437
• 发表时间: 2022-02-11
• 期刊: Science advances
• 影响因子: 13.6
• 作者: MacGregor-Chatwin C;Nürnberg DJ;Jackson PJ;Vasilev C;Hitchcock A;Ho MY;Shen G;Gisriel CJ;Wood WHJ;Mahbub M;Selinger VM;Johnson MP;Dickman MJ;Rutherford AW;Bryant DA;Hunter CN
• 通讯作者: Hunter CN

Structured Excitation Energy Transfer: Tracking Exciton Diffusion below Sunlight Intensity

• DOI: 10.1021/acsphotonics.4c00004
• 发表时间: 2024-02
• 期刊: ACS Photonics
• 影响因子: 7
• 作者: Guillermo D. Brinatti Vazquez;Giulia Lo;Gerfo Morganti;Cvetelin Vasilev;C. N. Hunter;N. V. Hulst

Supercharged PGR5-dependent cyclic electron transfer compensates for mis-regulated chloroplast ATP synthase

增压 PGR5 依赖性循环电子转移补偿错误调节的叶绿体 ATP 合酶

• DOI: 10.1101/2022.09.25.509416
• 发表时间: 2022
• 作者: Degen G