Electron transfer reactions keep living things alive: they underpin respiration, photosynthesis, metalloenzyme chemistry and protein folding. The Parkin group studies the biochemical reaction mechanism and biotechnological utility of electron transfer enzymes using electrochemistry, but this field of science is limited by an inability to "wire" any protein to any electrode. This project will develop a molecular biology and chemical biology method to site-specifically cross-link any protein to any conducting surface (Fascione group expertise). We will apply this method to gain new insight into bacterial protein folding mechanisms and fuel-producing enzymes.
The first test system will be a disulphide mediated protein folding enzyme from Mycobacterium tuberculosis. We will show that we can use a conductive surface to measure the rate of enzyme-catalysed protein folding as electrical current. We will probe the electron-transfer and proton-transfer pathway through the enzyme by making single site amino-acid exchanges near the Cys residues and measuring the change in the energetics (electrical voltage) and rate (electrical current) of disulphide bond formation. We will therefore understand the structure-function properties underlying the biochemical mechanism of protein folding. The binding affinity of the enzyme for different peptide substrates will also be quantified to understand the substrate specificity.
The second application of the protein wiring system will be to attach metalloenzymes such as hydrogenases to light-absorbing materials to achieve bio-catalysed solar-fuel production. We will thus showcase our "wiring" method as a way to develop new biotechnology.