Electron transfer in engineered single protein molecules

Lead Research Organisation: Cardiff University
Department Name: School of Physics and Astronomy

Abstract

All living organisms contain proteins - nanoscale molecular machines which have a myriad of functions. A large fraction of these proteins are "electron transfer" proteins which, as the name suggests, are capable of moving electrical charge from one place to another - either within the protein or between proteins. Such proteins are absolutely essential to the physics of life, controlling biological processes as varied as respiration, photosynthesis and the creation of organic molecules from basic elements (hydrogen, carbon, nitrogen, oxygen, etc.).

Although they actually function at essentially the single molecule level, most of our understanding of electron transfer (ET) proteins comes from experiments performed on large assemblies of protein molecules, not individual molecules. This is perhaps not surprising since it is usually difficult to locate a single molecule, or to obtain a measurable signal from just one molecule. Many traditional measurements therefore look at the optical properties of an assembly of molecules in solution. Others measure the electrical properties of metal surfaces covered in a layer of molecules.

The aim of our project is to develop a new way to measure individual ET protein molecules, and use these measurements to gain a better understanding of the ET process (directly relevant to theorists and a prerequisite for any biolectronic applications). To do this we first make two electrical contacts to the protein, and then incorporate it as part of an electrical circuit. By measuring how easy it is to pass current through the circuit, we can examine just how the protein functions to transfer electrons. We can also change other properties of the protein (such as a metal centre which is common in ET proteins) to examine their role in the ET process.

The first problem is how to make a reliable electrical contact to a single molecule. Fortunately, the methods already developed in protein engineering allow this to be done: it is possible to modify the protein surface to introduce specific chemical groups which strongly attach the molecule to a metal surface. This is achieved by altering the genetic material encoding the protein, so that the required chemical groups can be placed at precisely known positions in the protein. Multiple identical copies of the modified protein are produced in this way.

The second problem is how to examine just a single molecule. This has become possible over the past few years following the invention of the scanning tunnelling microscope or STM. This instrument allows an almost atomically-sharp metal tip to be brought close to a (sufficiently flat) metal surface; if the distance between tip and surface is small enough (around one nanometre - a millionth of a millimetre - or so) electrons in the tip can pass to the surface when a voltage is applied between them. The tip and surface don't have to touch, but the electrons pass because of the quantum mechanical "tunnelling" effect. By scanning the tip across the metal surface under computer control, it is possible to measure exactly how flat the surface is, and even form an image of individual metal atoms. If our protein molecules are sprinkled on the surface, it is possible to use the STM to see exactly where they have adhered, and to put the tip in contact with them. This completes our electrical circuit.

Measuring electron transfer through proteins in this way has not previously been done, and lets us explore the protein with a high degree of control. But it is not interesting simply for its own sake - it means we can better understand just how ET proteins operate at the level of a single molecule. Also, development of bioelectronic components using ET proteins, which is a subject of rapidly growing interest, ultimately depends on our ability to study them at the single molecule level and with electrical contacts.

Planned Impact

The impact on academic beneficiaries, including our collaborators, is described under the section "Academic Beneficiaries".

Science Made Simple (SMS) will be a core partner in the development of suitable outreach activity for us. They have a proven track of over eight years of engaging end-users (schools or public event beneficiaries) with contemporary research. They also have wide experience in training researchers in public engagement using a range of innovative formats such as performance skills, science busking and theatre production skills. Through training our research groups, they plan to create a legacy of skills amongst the research team that can be used for years to come as individual careers develop - benefiting especially the PDRA. SMS will also give advice in setting up web pages aimed at benefiting schools and the wider public. These pages will provide advice and offers of talks to schools.

SMS has provided a statement of support describing the benefits of this activity to them, and will also provide around £1.5k of work in kind (marketing and consultancy advice on targeting schools and other audiences, advice for PDRA and engagement events). SMS will benefit especially through the opportunity to work with cutting edge researchers to expand their knowledge around this area, which will benefit their outreach work with students and the public.

Communication by the applicants with the wider community has already been established through numerous talks to schools audiences and scientific societies. Cardiff University runs an open day every year and our research will be showcased in this forum. The School of Physics and Astronomy runs an annual 6th form conference where we shall continue to give presentations of our current research. The local Science Cafe in Cardiff, which any interested members of the local community may attend, will provide a valuable opportunity for public engagement.

One tool used in this project, synthetic biology, has been muh debated amongst politicians and the wider public. Policy makers need to know the worth, benefits and pitfalls (including public perception) in order to regulate the area; one of us (DDJ) recently (2009) reported to Defra on synthetic biology for policy makers. We also aim to interact with policy makers through the EPSRC, BBSRC or other research councils.

Staff employed on the grant will be trained in state-of-the-art technologies in (i) synthetic biology and (ii) single molecule studies. This will make them valuable to both the academic and private sector. Cardiff University runs an extensive staff development program with a wide variety of courses, ranging from research-related development to project management to leadership skills, which the PDRA will attend. Costs are covered by the Schools. The applicants will participate in the training in scientific methodology, outreach through SMS, and encourage the development of ideas and promote the PDRA's progression as an independent scientist.

The planned research is not directed at immediate technological application, but integration of protein molecules with graphene has a clear longer-term potential for novel routes to bioelectronic sensors, or even electronic control of biological events. We shall therefore actively explore avenues to apply the findings of our programme in this area. The procedures in place at Cardiff University ensure that research is constantly reviewed with the aim of identifying areas that merit patent protection. This works via the University's Research and Commercial Development (RACD) division who operate a Commercial Advisory Panel and who are are prepared to meet the costs involved. Commercial exploitation of our work would be of benefit to the researchers, Cardiff University and the UK technology base as a whole.

Publications

10 25 50

publication icon
Elliott M (2017) High-resolution electrochemical STM of redox metalloproteins in Current Opinion in Electrochemistry

 
Description We are investigating how single molecules of proteins transfer electrical charge. This involves both the measurement process itself, and the attachment of electrical contacts to the molecules.

So far, we have developed new ways of attaching contacts to a range of protein molecules so that we can contact not only with gold, but also (of great technological potential) with graphene.

We are now investigating the extrinsic factors (i.e. humidity and temperature) which influence the protein conduction using imaging of proteins on surfaces. This is done using a scanning tunnelling microscope, which allows us to image proteins through their electrical properties. We are also looking to develop useful devices by creating transistor devices with proteins as the active elements. The aim is to develop sophisticated single molecule detection systems with potential medical applications.
Exploitation Route We are developing devices, based on graphene transistors linked to proteins, for application to speedy detection of drug resistance in bacteria. This will be a collaborative venture involving bio- and medical scientists and physicists. One proposal has been submitted on real-time human growth hormone detection and another is in preparation. Linked to this, we are currently exploring collaboration with a group with expertise in carbon nanotube-DNA binding with the potential for a new type of sensor.
Further collaborative work has begun with Matteo Palma's group at Queen Mary University of London.
Sectors Creative Economy,Healthcare,Manufacturing, including Industrial Biotechology

 
Description Cardiff Synthetic Biology Initiative
Amount £45,908 (GBP)
Organisation SynbiCITE 
Sector Academic/University
Country United Kingdom
Start 05/2014 
End 09/2015
 
Description GW4 Accelerator award
Amount £71,189 (GBP)
Organisation GW4 
Sector Academic/University
Country United Kingdom
Start 03/2016 
End 10/2016
 
Description Iraqi HCED
Amount £88,000 (GBP)
Organisation Iraqi Government 
Sector Public
Country Iraq
Start 10/2015 
End 10/2019
 
Title Control of protein activity through bioconjugation and light. 
Description The incorporation non-natural amino acids (nnAA) using a reprogrammed genetic code always new chemistry to be incorporated into proteins that can be used to modulate activity. We have used phenyl-azide chemistry as means to implement our strategy through the incorporation of the nnAA p-azido-phenylalanine. Two chemistries are available to control protein activity: (1) covalent bioconjugation with small non-biological chemical adducts; (2) irradiation to form a reactive nitrene radical that can follow different chemical routes. Both induce local conformational changes in the protein that can either up or down regulate protein activity. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact 1. We have now shared our plasmids with other groups around the world who wish to use our approach. 2. We now have several collaborations which include the exchange of researchers aimed primary at using the bioconjugation approach. 
 
Title Direct protein interfacing with carbon nano materials and DNA tiles. 
Description By incorporating non-natural amino acids into a protein at defined positions we can now precisely control assembly of hybrid protein materials. We have so far demonstrated the approach with two systems: (1) site-specific attachment of ssDNA to provide addressable assembly on DNA origami tiles. Using a non-biological reaction handle incorporated using a reprogrammed genetic code, ssDNA can be site-specifically attached to the protein of interest (POI) at an optimal position. The hybrid ssDNA-protein system can then be assembled on base DNA tiles with the ssDNA acting as the addressable element and the protein the active component. We have used this system to assemble multiple proteins on a single tile. When assembled, the proteins can show enhanced activity. (2) site specific attachment of proteins to carbon nano materials such as single walled carbon nanotubes (swCNTs) and graphene. Using a non-biological reaction handle incorporated using a reprogrammed genetic code, we have successfully attached proteins to these carbon nanomaterials to get functional communication between proteins. We have achieved this is two ways. (i) attachment of conjugates that allow non-covalent interfacing with the pi electron system of the carbon nanomaterials. (ii) direct covalent attachment using light activated chemical groups incorporated into the protein. We assembled, we have seen communication between the protein and carbon nano materials. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact 1. New ways of working. 2. Collaborations with other institutions including University of Southampton, Manchester and Queen Mary. We are jointly exploring how to exploit the new approach to nanoassembly. 
 
Title New approaches to designed protein oligomerisation. 
Description One of the most important events a protein will undergo is associating with itself or other proteins to from a functional complex; this is known as oligomerisation. Protein oligomerisation is common place in nature, with the majority of cellular proteins existing either permanently or transiently as oligomers. Oligomerisation is normally cooperative and synergistic in that properties such as function and stability are greatly enhanced compared to the monomeric form, and new properties can emerge (e.g. functional enhancement, switching); there is normally communication between individual monomer units that leads to these new or enhanced properties. The method involves taking normally monomeric proteins and then designing, building and testing new oligomeric protein species. While oligomerisation could be considered desirable, it is difficult to engineer into functional monomeric proteins due to the complexity of natural protein-protein interfaces. To meet this challenge, we used a new synthetic biology approach for generating protein oligomers: biorthongonal Click crosslinking using a reprogrammed genetic code to incorporate non-natural amino acids (nnAA) at designated residues in a target protein. At least two different types of nnAA were used that are not found in nature but can react on a 1 to 1 basis to form a defined crosslink. Using this system, we can explore the construction of dimer, trimers and beyond composed of identical and mixed protein units to generate a myriad of new structures of potential fundamental and technological use. To date the research has focus on autofluorescent proteins (e.g. GFP) but will quickly move on to constructing multi-enzyme complexes. This interdisciplinary project has additional focus on in silico protein design and engineering (e.g. protein docking simulations) and combines elements chemistry and biophysics. Techniques that embrace the overall concept. Primary: computational analysis, synthetic biology (reprogrammed genetic code systems). Secondary associated techniques: molecular biology (cloning and mutagenesis), protein chemistry (purification and analysis), protein 3D structure determination, biophysical analysis (various spectroscopy methods, including single molecule analysis). 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact This is an nascent project with the aim of generating communicating protein complexes akin to those found in nature. Our early designs and constructed systems show great promise including new protein structural scaffolds, functional synergy and new emergent functions. 
 
Title STM temperature and humidy control system 
Description Developed a home built STM with high stability control of temperature (30 to 0 degree C) and humidity (100% to <1% relative humidity). 
Type Of Material Improvements to research infrastructure 
Provided To Others? No  
Impact Two publications being prepared. Additionally, a joint research project (with Chemistry Depart, University of Liverpool) is in progress and should lead to a new publication. Will then offer facility to other researchers. 
 
Title Site-specific protein modification through the use of non-natural amino acids. 
Description We have used expanded genetic code systems to incorporate new chemistry not present in nature with the aim to link protein to non-biological components ranging from small molecules to materials to other biomolecules. We have focused primarily on the use of phenyl azide chemistry to: (1) photo control protein activity; (2) link proteins to oligonucleotides for bottom-up assembly; (3) add small molecule adducts that regulate protein activity; (4) link proteins to materials such as graphene and carbon nanotubes. 
Type Of Material Biological samples 
Year Produced 2012 
Provided To Others? Yes  
Impact We have used the approach to demonstrate the core aspects above namely (1) photo control protein activity; (2) link proteins to oligonucleotides for bottom-up assembly; (3) add small molecule adducts that regulate protein activity; (4) link proteins to materials such as graphene and carbon nanotubes. As stated above. 
 
Description Designed protein-carbon nanotube interfacing. 
Organisation Queen Mary University of London
Department School of Biological and Chemical Science QMUL
Country United Kingdom 
Sector Academic/University 
PI Contribution We have engineered a series of proteins ranging from autofluorescent proteins to antibiotic inhibiting proteins to contain a new reaction handle at specific sites. This new reaction handle, azidophenylalanine (azF) can used to interface proteins with CNTs using Click Chemistry.
Collaborator Contribution The collaborator, Dr Matteo Palma, is an expert in CNTs and their use including in terms of their application to sensing. They provide the CNT materials and analysis approaches to the project.
Impact 1 paper: see Freeley, Worthy et al in the main list. 1 grant in preparation: we plan on submitting a grant concerning the construction of novel biosensors. Multidisciplinary: the discipline involved include synthetic biology, protein engineering and design, biochemical/biophysical analysis, nanoscience, materials science, chemistry and physics.
Start Year 2016
 
Description Designed protein-nano carbon and protein-protein interfacing. 
Organisation Cardiff University
Department School of Pharmacy and Pharmaceutical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have generated engineered variants of fluorescent proteins that can be photochemically linked to nano-carbon or linked together to generate novel communicating oligomers.
Collaborator Contribution The partners, namely Dr Oliver Castell, is an expert in single molecule imaging using total internal resonance fluorescence microcopy (TIRFM). Through our collaboration, Dr Castell has undertaken extensive experimental measurement and analysis of the data as a contribution to the project.
Impact Outputs: 1 paper submitted and 1 paper manuscript in preparation. This is multidisciplinary project involving protein engineering and synthetic biology together with physics (single molecule measurements and analysis)
Start Year 2017
 
Description Protein engineering for new biosensor implementation. Director of Synthetic Biology. 
Organisation Molecular Warehouse Ltd
Country United Kingdom 
Sector Private 
PI Contribution The research background of myself (Dr Dafydd Jones) has mean that a small start-up company, Molecular Warehouse, are invited me to be their Director of Synthetic Biology.
Collaborator Contribution Due to the commercially sensitive nature of this work, I will not state any details. The partners, Molecular Warehouse, wish me to join their company on a part-time basis initially as their director of synthetic biology. This is primarily to lead their protein engineering and production efforts.
Impact There are no outputs as of yet
Start Year 2017
 
Description Invited speaker at conferences. These include RSC Chemistry in the New World of Bioengineering and Synthetic Biology; RSC Chemistry and Biology Interface; RSC Bioorganic Firbush Meeting: Protein engineering and directed evolution; Synthetic biology in Pharma; European Society of Bio-organic Chemistry meeting; SWSB Structural Biology meeting; SCN Synthetic Biology symposium; Zing International Structure Biology and Drug discovery conference; Synthetic Biology Symposium in Glasgow; Harden Confer 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I have talked at many different conferences presenting work related to the funded work.

Making contacts and advertising the work supported by the grants.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013
 
Description Invited talks at University of West Virginia USA, Modena Italy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Raised group profile and international contacts.
Year(s) Of Engagement Activity 2013
 
Description Lecture on Nanoscience (University of Sarajevo) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact On 16th April 2015, 120 undergraduates, postgraduates and general public attended a lecture on Nanotechnology at the University of Sarajevo. Local TV journalists attended and it led to an interview broadcast on Bosnian State Television.
It also led to discussion of possible research involvement in nanoscience in a university setting where little dedicated equipment exists.
Year(s) Of Engagement Activity 2014,2015
 
Description School Visit (Wales). These include (i) Wales Gene Park sixth form conference (Nov 2012) to ~1600 A- level students at St David's Hall, Cardiff; (ii) visit to local sixth form to discuss genetic modification; (iii) visit to local primary school to talk and demonstrate why proteins are useful (e.g. washing powders and cheese making). (iv) talk at Coleg Gwent to A-level students concerning synthetic biology. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact talk and discussion with students

Informed students on current science
Year(s) Of Engagement Activity 2009,2015
 
Description Talks at Universities. These include the following Bristol, Cambridge, Kent, Liverpool, Nottingham (x2), Oxford, Reading, Sheffield, Warwick (x2) and Cardiff University. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Talks to students, PhDs, postdocs and PIs in areas related to those associated with the grants.

Collaborations.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013
 
Description Talks at universities (Birmingham, Exeter, Liverpool, Nottingham, Cardiff) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact University or conference talks.
Year(s) Of Engagement Activity 2013,2014,2015