Award details

Quantification of the forces that mediate electron transfers between proteins

Reference	BB/P002005/1
Principal Investigator / Supervisor	Professor Matthew Johnson
Co-Investigators / Co-Supervisors	Krebs Professor of B Christopher Hunter, Dr Cvetelin Vasilev
Institution	University of Sheffield
Department	Molecular Biology and Biotechnology
Funding type	Research
Value (£)	380,814
Status	Completed
Type	Research Grant
Start date	01/01/2017
End date	31/12/2019
Duration	36 months

Abstract

In this project we will quantify the transient interactions that sustain biological electron transfers between small redox diffusible proteins and larger membrane bound complexes. Such forces must be transient yet highly specific to allow the electron transfer chains in respiration and photosynthesis to turnover rapidly so they efficiently produce ATP. Previously these interactions have been analyzed in bulk solution by absorption spectroscopy; now we have developed a new nanomechanical mapping application of atomic force microscopy that allows us to quantify at the single molecule level the interaction forces and binding probabilities between electron transfer donor and acceptor proteins. These new measurements will provide new insights into the formation of a transient interface that brings the reduced and oxidised cofactors into proximity for electron transfer, and the way that this interface changes in order to allow separation of the proteins. Our preliminary data have highlighted the power of our AFM approach by uncovering new and unexpected features of biological electron transfer reactions, such as their redox dependency and the post-electron transfer reorganization of the binding interface occurring on a timescale of hundreds of mircoseconds that leads to dissociation. Our aim now is to use this new technique to fully understand the nature of the interactions that govern the ET complex formed between the cytochrome c2 -photosynthetic reaction centre pair from the bacterium Rhodobacter shaeroides and the plastocyanin-photosystem I pair from the green algae Chlamydomonas. The results will bring a fresh perspective to understanding biological electron transfer that will be widely applicable in bacteria, chloroplasts and mitochondria.

Summary

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.

Impact Summary

Economic impacts: 1. Our collaborative relationship with Bruker involves the testing of batches of their latest probe designs to assess their performance for high-resolution nanomechanical and topological imaging of biological membranes. This in-kind benefit that we obtain from our close relationship with Bruker has been established over many years and is worth ~£10,000. The feedback we provide directly informs Bruker's probe manufacturing process and promises to enhance the resolution routinely available to other biological users through improved probe sharpness. Bruker are also interested in our unique development of their PF-QNM AFM technique, 'affinity-mapping AFM' which underpins this project and our recent work. Bruker believe that the electron transfer problem we have identified, with forces in the range of 50-750 pN and occurring on a timescale of microseconds to milliseconds, provides the ideal experimental system to fully explore the capabilities of PF-QNM. To this end Bruker engineers involved in AFM development will visit Sheffield to meet and discuss the requirements our work places on their AFM system, discuss our suggestions for improvements to the technology and advise on how we can further our experiments with the latest developments. Bruker will use the data from our published experiments to advertise the capabilities of the new instrument to other customers worldwide. 2. The wider biology research community within the UK will benefit from our development of nanotechnological tools such as PF-QNM atomic force microscopy for imaging and functional measurements of biological samples. Such developments are crucial to solidifying the UK's position as a leader in bionanotechnology research and will directly impact upon other research fields such as medicine and disease (e.g. studying cell membranes of bacterial and protozoal pathogens to identify novel drug targets). 3. Understanding the natural principles that underpin efficient electron transfer can be used by future commercial entities that wish to develop artificial and biomimetic systems that harvest, convert and store solar energy and capture carbon dioxide from the atmosphere. Such solar and carbon capture devices of the future will impact upon the wealth and health of the UK in the future by reducing carbon emissions and potentially ameliorating environmental change by increasing the fraction of green energy we produce as a nation. 4. Commercial projects to exploit photosynthetic microalgae for the production of biofuels will be able to build on our development of bionanotechnological techniques to simultaneously investigate structure and function in biomembranes. These novel methods could be used to explore the effects on photosynthetic membrane organisation of altered metabolism in biofuel-producing strains of the green algae Dunaliella and Nannochloropsis. The potential impact of the research in the selection and development of algal strains for biofuel production will mean that the UK will be less dependent in the future on fossil fuels as sources of electricity and transport fuel. 5. Agro-biotech companies such as Syngenta are developing mutants of rice to create future commercial opportunities for increasing the productivity of crops by manipulating electron transport. Understanding the kinetic bottlenecks in electron transport could provide new targets for genetic manipulations of photosynthesis by Syngenta, potentially creating crops with enhanced productivity, increasing global food security. 6.The technical approaches used in this research programme benefit UK society by providing training and experience in a multidisciplinary array of microscopic, spectroscopic and biochemical techniques and other practical techniques. Such skills provide the basis for innovative, cross-discipline solutions to crucial biological and environmental problems of the present and future and will play a part in developing a successful bio-based economy.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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