Quantification of the forces that mediate electron transfers between proteins

Lead Research Organisation: University of Sheffield
Department Name: Molecular Biology and Biotechnology

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description We have established a new method, based on atomic fore microscopy, for investigating the interactions between electron transfer proteins involved in the live-giving reactions of photosynthesis that sustain plants, algae and certain bacteria.

Our investigations allowed us to quantify for the first time the transient forces that mediate these interactions, understand their temporal evolution and their dependence on the environmental state.

For electron transfer to work efficiently the proteins involved must come together specifically and quickly yet also the interaction must be sufficiently transient to allow multiple turnovers per second. Our results show that nature has achieved this by ensuring only proteins in opposite electron transfer states bind one another, similar to how the opposite poles of a magnet attract. This 'redox-gating' ensures that the productive interactions are favoured and unproductive interactions are avoided increasing the efficiency of photosynthesis.

To achieve a molecular level of understanding of one of the key electron transfer proteins involved in photosynthesis (cytochrome b6f) we determined its high-resolution atomic structure by cryo-electron microscopy. This breakthrough has allowed us to deepen our knowledge of how the central electrical connection in photosynthesis works, an unexpected but very positive outcome of the grant that was published in Nature and featured in Scientific American.

The aim now is to take forward our study of electron transfer protein using this method to understand if similar rules apply to those complexes involved in respiration that sustain animals and bacteria.
Exploitation Route Our research is relevant to 1) Bioenergy and 2) Synthetic biology aspects of BBSRC strategy, and has provided fundamental knowledge key to understanding photosynthesis at the molecular level, revealing bottlenecks in the electron transfer systems that may provide future genetic targets to improve the efficiency of such processes in plants, bacteria and algae for growth of higher yield crops, biofuels, medicines or other commercially valuable products in cell factories. Our work also encompasses 3) Technology development for the biosciences - the development of our method to quantify transient protein-protein interactions provides a new, widely applicable tool (a) for characterising events at membrane interfaces such as ligand-receptor interactions involved in disease and (b) for mapping the spatial location of protein complexes within a membrane1
Sectors Energy,Pharmaceuticals and Medical Biotechnology

URL https://www.scientificamerican.com/article/key-photosynthesis-complex-viewed-in-spinach/
 
Description Our collaborative relationship with Bruker (the manufacturer of the atomic forces micropsope) involved the testing of batches of their latest probe designs to assess their performance for high-resolution nanomechanical and topological imaging of biological membranes. 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. This impact of our work was delivered across months 1-36 of the project with the PDRA testing the beta probes alongside the existing commercially available probes during the course of the project. Bruker were 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 in involved in AFM development visited 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. This impact was delivered with visits by Bruker Engineers to Sheffield at the end of years 1,2 and 3 of the project, which will be feedback to into ongoing AFM development at their Santa Barbara facility in California. Bruker will use the data from our published experiments to advertise the capabilities of the new instrument to other customers worldwide. This impact of our work is delivered as papers using PF-QNM are published in international journals during the course of the project, which Bruker are able to refer other customers too assisting them to sell more instruments.
First Year Of Impact 2018
Sector Digital/Communication/Information Technologies (including Software),Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title Affinity-mapping Atomic force microscopy 
Description We have developed a single molecule technique that provides a new level of information on the dissociation of ET complexes, which utilises the Peak-Force quantitative nanomechanical mapping (PF-QNM) mode of our atomic force microscopes (AFM). We can quantify the interaction forces, binding probabilities and dissociation rates of transient Electron transfer complexes at the single-molecule level on timescales from 50┬Ás to 100 ms, both in vitro and at the membrane surface. This method can also be used to quantify transient protein-protein interactions at membrane interfaces such as ligand-receptor interactions and for mapping the spatial location of protein complexes within a membrane. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact Bruker (manufacturer of atomic fore microscopes) are 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 in involved in AFM development visited 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. Publciatiosn arising form our work may generate impact in terms of new sales and technology/ software development for Brukers AFM systems.