Single protein crystal spectroscopy and crystallography of hydrogenase under electrochemical control

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Proteins are the nanoscale 'machines' that control almost all processes in cells. Importantly they are responsible for speeding up (catalysing) chemical reactions that make the essential molecules for life and release or store energy. Understanding the structures of individual proteins at the atomic level has been absolutely key in building up our understanding of how they contribute to the function of cells. In particular, the technique of X-ray crystallography has been extremely valuable in providing 'snapshot' images of many different proteins at the level of individual atoms. In this approach, crystals of the protein are prepared, and are probed using a high energy X-ray source which diffracts offindividual atoms in the crystal, giving a pattern that reveals the positions of atoms in the protein. A series of these 'snapshots' is needed to understand each step in how a protein works, and it is often difficult to trap proteins in specific states relevant to their function in order to obtain a complete set of 'snapshots'. This is particularly true for a group of 'redox' proteins which catalyse chemical reactions involving the transfer of electrons - oxidation and reduction reactions. A further challenge arises because these redox proteins often incorporate metal atoms that are susceptible to damage by X-rays during the data collection for crystal structure determination. It is very timely that we find new tools for studying these sort of proteins, because they catalyse many chemical reactions which are relevant to solving big global challenges, including how to use hydrogen as a sustainable fuel, how to capture the greenhouse gas carbon dioxide and turn it into useful chemicals, and how to efficiently produce the fertilisers needed to sustain the world's growing population. In this project, we demonstrate a completely new approach to controlling and verifying the state of redox proteins that will allow crystal structure snapshots to be produced for many more of the important functional states of these redox proteins. Our focus is a protein called hydrogenase which allows microbes to live on hydrogen gas as their energy source. In earlier preliminary work, we have shown that we can use electrodes to control a single crystal of hydrogenase to generate uniform states relevant to its function. This provides an unprecedented way to get proteins in single crystals into specific states ready to record X-ray crystallographic 'snapshots'. At the same time, we make use of imaging using infrared light with a special infrared microscope to confirm the state of the protein in the crystal. During the project, we will show that we can prepare specific states of protein crystals in this way and then record their X-ray structures to yield snapshots of previously unseen states of the protein. This will yield new information on how proteins function as efficient catalysts for the important reactions mentioned above. We will also use the infrared imaging approach to check the crystals after exposure to X-rays to make sure that the state of the protein in the crystal has not been damaged during X-ray crystallographic data collection. This approach will lead to much more reliable snapshots of redox proteins.The project thus represents a step change for structural biology of redox proteins and understanding the function of proteins which may teach us how to solve important global problems. We would like these tools to become widely available to structural biologists who solve the structures of complicated proteins, and during this project we aim to develop our approaches so that they can be readily implemented.What we learn about the way that hydrogenases work in the course of this project will help to propel biotechnological applications of hydrogenases, and will underpin development of alternative chemical catalysts based on the cheap metals, nickel and iron, found inside the hydrogenases.