Chemical Biology to Wire Enzymes to Electrodes for Biotechnology Applications

Lead Research Organisation: University of York
Department Name: Chemistry

Abstract

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.

Publications

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Yates NDJ (2018) Methodologies for "Wiring" Redox Proteins/Enzymes to Electrode Surfaces. in Chemistry (Weinheim an der Bergstrasse, Germany)

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 30/09/2015 29/09/2023
1792687 Studentship BB/M011151/1 30/09/2016 30/03/2021 Nicholas David Yates
 
Description A system has been developed whereby a set of molecules called "hydroxylamines" can be immobilised onto an electrode surface (a conductive surface). These molecules can be used to bond to the sugars found on the surface of interesting proteins, immobilising the proteins onto the conductive surface. With a bit of luck, and the right protein, this allows the conductive surface to pass electricity to the immobilised protein. Some proteins can then use this electricity to do a chemical reaction. This sort of technology is widely used in medical applications in "biosensing," the most famous example being the glucose oxidase biosensor diabetics use to monitor their blood glucose levels. A lot of mammalian proteins have sugars on their surface, so the hydroxylamine-mediated immobilisation methodology is potentially usefull.

Additionally the use of a class of molecules called "triazabutadienes" in surface functionalisation of conductive surfaces has been explored. This is a literature first, and while further experiments are pending, we hope to be able to use these molecules to immobilise proteins directly onto conductive surfaces, but attaching the triazabutadiene to the protein first, then getting the triazabutadiene into a reactive state using a pulse of UV light.

Additionally, experiments have been conducted whereby interesting fungal enzymes are immobilised onto conductive nanotubes. These enzymes are interesting as they can be used in biofuel production, and although people know they work we aren't sure how they work. The experiments that we are currently conducting could unlock the secrets of these enzymes, allowing us to design chemicals that can be used to make biofuel.
Exploitation Route Others will use the techniques I have developed to study interesting enzymes and proteins.
Sectors Chemicals,Electronics,Energy,Pharmaceuticals and Medical Biotechnology

URL https://pubs.acs.org/doi/abs/10.1021/acs.langmuir.9b01254