Final Report Summary - MAGIC (MAGnetic Innovation in Catalysis)
The MAGnetic Innovation in Catalysis (MAGIC) network is a Marie Curie Initial Training Network (ITN) project funded by the European Union 7th Framework Programme (2013 PEOPLE Work Programme).
The aim of the MAGIC ITN was to train the future generation of leading of biological catalysis and enzymology investigators through the development of new enabling technologies that will advance the physical understanding of catalysis and mechanism. Research has shown that enzyme catalysts exploit the coupling of motions to the reaction coordinate and employ classical and quantum effects (e.g. quantum tunnelling) in their reaction cycles. Roles for quantum entanglement and spin chemistry are also suggested. Prompted by these discoveries, many groups are currently focusing their research on this field, and this offers great career opportunities. The aim was to understand the physical and chemical basis of enzyme catalysis and exploit this information in predictive design, thereby underpinning the exploitation of enzyme catalysts in industrial biotechnology, manufacture and diagnostics. There is a need for outstanding young scientists to pursue collaborative research projects in which the mechanistic details of enzyme systems can be explored using new experimental capabilities to probe the contributions of motions across multiple spatial and temporal timescales and quantum chemical effects. The projects were based upon innovative, versatile and unique experimental techniques. Their scientific focus was on the physics of magnetic resonance derived from original concepts of the participating senior scientists. The new methods can be applied to a range of important biological catalysts to probe the mechanistic importance of coupled motions and quantum physico-chemical effects.
The MAGIC network coordinator is Professor Nigel Scrutton from the Manchester Institute of Biotechnology, the main ITN beneficiary being the University of Manchester (www.manchester.ac.uk). The MAGIC ITN consortium comprises of 11 original associated partners from academia, small-to-medium-sized enterprises (SMEs) and industry and one further partner (CR UK) was added during the project lifetime in 2016:
1) University of Copenhagen, Denmark, Dr Stergios Piligkos (www.ku.dk)
2) University of Joseph Fourier, France, Professor Martin Blackledge (www.ujf-grenoble.fr)
3) University of Lund, Sweden, Professor Mikael Akke (www.lunduniversity.lu.se)
4) University of Freiburg, Germany, Professor Stefan Weber (www.uni-freiburg.de)
5) University of St Andrews, UK, Dr Janet Lovett (www.st-andrews.ac.uk)
6) University of Tokyo, Japan, Professor Jonny Woodward (www.u-tokyo.ac.jp)
7) C4x Discovery, UK, Charles Blundell (www.c4xdiscovery.com)
8) SARomics, Sweden, Dr Björn Walse (www.saromics.com)
9) TgK Scientific, UK, Mr Ted King (www.hi-techsci.com)
10) AstraZeneca, UK, Kevin Embrey (www.astrazeneca.com)
11) Bruker, Germany, Dr Alistair Fielding (www.bruker.com)
12) Cancer Research UK, UK, Dr Ian Waddell (www.cancerresearchuk.org)
All 12 Early Stage Researchers (ESRs) were recruited and they each completed their three-year research contract. Ten were from the EU (two from each of Poland, Romania and Spain; one from Germany, Italy, Portugal and Croatia), two were from non-EU countries (one from each of Kazakhstan and USA) and overall 75% of the ESRs were women. The initial focus was on general student induction, health & safety training, early scientific & technical training, writing individual literature reviews, starting their research activities and presenting progress at various meetings. In the ESRs’ second year the focus was on continued progression of their PhD projects along with additional training, secondments and attendance at workshops and conferences. In the final year of their PhD most of the ESRs had to focus on delivering data for their thesis; as it was a challenge to do all the training required for these ITN cross-disciplinary projects as well as spend enough time actually producing sufficient high-quality results for their PhD. As planned, at the end of the project in May 2018, the MAGIC ITN had implemented the total expected 432 research months. All 12 ESRs were registered for a PhD at the University of Manchester and at the time of the report ten were continuing to write up and two had completed their PhD. The project was managed through regular meetings that covered the scientific research, the training of the individual ESRs, the involvement of the ITN partners and the outreach activities.
The project objectives were split into four scientific work packages. The first of these aimed to develop the application of high-pressure nuclear magnetic resonance (NMR) measurements for the elucidation of the structure, electronics, and dynamics of enzymes. The second work package was to develop advanced experimental capabilities using time-resolved electron paramagnetic resonance (EPR) (also known as ESR - electron spin resonance) to understand the dynamics of complex biological redox reactions. The objective of the third work package was to exploit time-resolved magnetic field effects (MFEs) to develop a range of new experimental capabilities for the study of short-lived radicals. The final scientific work package aimed to develop new cryo-EPR spectroscopy capabilities and to exploit these approaches to identify very early intermediates and the quantum behaviour of enzyme catalysts. An additional major objective of the MAGIC ITN was the training of 12 ESRs in these scientific areas.
The research carried out for the first work package included the development of expression and purification protocols to provide unlabelled and isotopically labelled pentaerythritol tetranitrate reductase (PETNR), labelled catechol-O-methyl transferase (COMT) and human phosphoglycerate kinase (hPGK). Subsequently backbone and side-chain assignments was completed for these proteins using NMR spectroscopy enabling further NMR spectroscopic investigation to further understand these enzymes and to develop new catalytic models. For the second work package a new flow-flash EPR instrument was designed, optimised and then tested using methylcobalamin, protochlorophyllide-containing systems. Separately, caged molecules were prepared for laser de-caging enzymatic studies and test systems were trialled on a new freeze-quench instrument that was developed in conjunction with MAGIC ITN partner TgK Scientific. Additionally, full-length nitric oxide synthase (nNOS) and labelled calmodulin (CaM) were prepared and significant progress was made by studying these using a variety of EPR techniques. As part of the third work package a novel transient absorption instrumentation was developed to enable investigations of radical pair magneto-sensitivity across all various timescales for chemical and biological systems. For this research the substrate Pchlide, the deuterated cofactor NADPD, the wild type protochorophyllide oxidoreductase (POR), wild type ethanolamine ammonia lyase (EAL) and selected enzyme mutants were produced. A number of enzymatic studies were carried out with these enzymes including enzyme kinetic characterisation and MFE studies are ongoing. For the fourth work package a new EPR spectroscopy cryostat was designed, built and then tested down to below 300 mK (milli-Kelvin) using model systems and a calibrated ruthenium(IV) oxide thermometer. Based on this, future cryo-EPR systems will be designed and optimised to achieve even lower temperatures and to allow light irradiation of the samples. Various chemical and biological systems were studied using low temperature EPR studies and these low temperature EPR spectra were simulated using different software packages. Additional cryo-EPR research was carried out on lanthanide-series complexes, cobalamins and Pchlide.
The main scientific results include the delivery of four new instrument capabilities, access to various biological proteins and a large amount of scientific data and understanding based on the research already carried. out. The instrument capabilities included the development and testing of a cryogenic probe for low temperature (down to less than 1 Kelvin) electron paramagnetic resonance (EPR) measurements. These measurements enable the study and identification of very early intermediate transition states along a reaction coordinate as the extremely low temperatures ‘freeze out’ the nuclear motions and slow reaction paths so that electronic transitions and the quantum behaviour of enzyme catalysts can be studied. Secondly, a continuous flow electron paramagnetic resonance (EPR) apparatus has been made that enables replacement of reactants within the resonator of the EPR instrument allowing accumulation of the signal derived from the reaction paramagnetic species. The data generated provides insight into reaction mechanism research for systems where it is challenging to study short-lived radicals at room temperature because of their transient nature. Thirdly, a new 'flash-smash' sample preparation instrument for time-resolved electron paramagnetic resonance (EPR) has also been build, tested and used with stable 'caged' precursors to generate radicals and reaction intermediates by laser photolysis and trap these at low temperature to enable spectroscopic studies. The fourth instrument capability is a newly developed state-of-the-art variable magnetic field instrumentation for transient absorption measurements that enables magnetic field effect investigations of chemical and biochemical systems on the ultrafast time scales (from femtoseconds to seconds). Various wild-type and mutant enzymes have been prepared, characterised and studied as part of the ITN and these will be used to provide further enzymatic understanding and they have the potential for application in synthetic biology. In addition, wild-type and mutant cryptochromes have been prepared and studied to provide new understanding of their role and they have potential applications in photo-biology and optigonetics. The large amount of scientific data produced as part of the ITN has been used to advance enzymatic and catalytic understanding and more of this will be made available in future publications.
A major component of the MAGIC ITN was the training provided to the 12 ESRs. Initial training focused on general student induction activities, health & safety training, and early scientific training. Subsequently, all the ESRs have had more detailed technical training; attended various lecture courses and symposia; given various presentations on their work; been on initial secondments to the ITN partners; written a literature review for their individual scientific projects; written up a first year report and then passed a first-year viva by independent academics. They have also attended a variety of internal, national and international workshops and conferences; as well as having the opportunity to undertake a range of broader skills training as part of delivering their individual career development plans. Overall, the MAGIC ITN delivered most of the training and the scientific objectives.
A wide range of dissemination activities has taken place in order to communicate to multiple audiences. Throughout the duration of the ITN a dedicated website (www.magic.manchester.ac.uk) has been maintained. This website explains the objectives, details the researchers, highlights the scientific background, and reported on the progress, publications, presentations and other news highlights. Publication in peer-reviewed journals was a key dissemination activity for the network. To date 12 publications have been accepted and a number of these were published in high-impact journals such as “Nature Chemistry”, “Angewandte Chemie” and “Journal of the American Chemical Society”. Another key activity was the presentation of the research at multiple workshops and conferences. To date, over 40 poster presentations and over 50 oral presentations have been given at local, national and international conferences. These included presentations from all 12 of the Early Stage Researchers and from all 4 scientific work packages of the ITN. As part of our outreach activities to the wider scientific community and public, the Early Stage Researchers have been helped run four annual Manchester Institute of Biotechnology Open Days for senior school pupils, been involved in science festivals, participated in over 40 activities/events with school children, and several ‘question and answer’ scientific sessions. Finally, and number of the academics have made YouTube videos explaining their research that are openly available on the internet (https://www.youtube.com/channel/UCXf3CFkyGpa2eIeT5wz_Prw).
The MAGIC award delivered new science and technology capabilities that has and will advance knowledge of the detailed mechanisms of enzyme catalysts. On the longer term, deeper appreciation of enzyme mechanisms through implementation of these innovative science and technology developments will transform understanding of basic catalytic mechanisms. This will aid in the predictive design/redesign of catalysts e.g. for wider applications in the bioeconomy. The award has trained 12 ECRs who are trained to work at the interface of catalysis science and major economic growth areas for the EU including advanced materials, chemicals and biotechnology. MAGIC has therefore contributed to the structuring of an informed workforce and new scientific capabilities that will enable the EU to benefit from opportunities in catalysis science relevant to economic growth and quality of life.
The aim of the MAGIC ITN was to train the future generation of leading of biological catalysis and enzymology investigators through the development of new enabling technologies that will advance the physical understanding of catalysis and mechanism. Research has shown that enzyme catalysts exploit the coupling of motions to the reaction coordinate and employ classical and quantum effects (e.g. quantum tunnelling) in their reaction cycles. Roles for quantum entanglement and spin chemistry are also suggested. Prompted by these discoveries, many groups are currently focusing their research on this field, and this offers great career opportunities. The aim was to understand the physical and chemical basis of enzyme catalysis and exploit this information in predictive design, thereby underpinning the exploitation of enzyme catalysts in industrial biotechnology, manufacture and diagnostics. There is a need for outstanding young scientists to pursue collaborative research projects in which the mechanistic details of enzyme systems can be explored using new experimental capabilities to probe the contributions of motions across multiple spatial and temporal timescales and quantum chemical effects. The projects were based upon innovative, versatile and unique experimental techniques. Their scientific focus was on the physics of magnetic resonance derived from original concepts of the participating senior scientists. The new methods can be applied to a range of important biological catalysts to probe the mechanistic importance of coupled motions and quantum physico-chemical effects.
The MAGIC network coordinator is Professor Nigel Scrutton from the Manchester Institute of Biotechnology, the main ITN beneficiary being the University of Manchester (www.manchester.ac.uk). The MAGIC ITN consortium comprises of 11 original associated partners from academia, small-to-medium-sized enterprises (SMEs) and industry and one further partner (CR UK) was added during the project lifetime in 2016:
1) University of Copenhagen, Denmark, Dr Stergios Piligkos (www.ku.dk)
2) University of Joseph Fourier, France, Professor Martin Blackledge (www.ujf-grenoble.fr)
3) University of Lund, Sweden, Professor Mikael Akke (www.lunduniversity.lu.se)
4) University of Freiburg, Germany, Professor Stefan Weber (www.uni-freiburg.de)
5) University of St Andrews, UK, Dr Janet Lovett (www.st-andrews.ac.uk)
6) University of Tokyo, Japan, Professor Jonny Woodward (www.u-tokyo.ac.jp)
7) C4x Discovery, UK, Charles Blundell (www.c4xdiscovery.com)
8) SARomics, Sweden, Dr Björn Walse (www.saromics.com)
9) TgK Scientific, UK, Mr Ted King (www.hi-techsci.com)
10) AstraZeneca, UK, Kevin Embrey (www.astrazeneca.com)
11) Bruker, Germany, Dr Alistair Fielding (www.bruker.com)
12) Cancer Research UK, UK, Dr Ian Waddell (www.cancerresearchuk.org)
All 12 Early Stage Researchers (ESRs) were recruited and they each completed their three-year research contract. Ten were from the EU (two from each of Poland, Romania and Spain; one from Germany, Italy, Portugal and Croatia), two were from non-EU countries (one from each of Kazakhstan and USA) and overall 75% of the ESRs were women. The initial focus was on general student induction, health & safety training, early scientific & technical training, writing individual literature reviews, starting their research activities and presenting progress at various meetings. In the ESRs’ second year the focus was on continued progression of their PhD projects along with additional training, secondments and attendance at workshops and conferences. In the final year of their PhD most of the ESRs had to focus on delivering data for their thesis; as it was a challenge to do all the training required for these ITN cross-disciplinary projects as well as spend enough time actually producing sufficient high-quality results for their PhD. As planned, at the end of the project in May 2018, the MAGIC ITN had implemented the total expected 432 research months. All 12 ESRs were registered for a PhD at the University of Manchester and at the time of the report ten were continuing to write up and two had completed their PhD. The project was managed through regular meetings that covered the scientific research, the training of the individual ESRs, the involvement of the ITN partners and the outreach activities.
The project objectives were split into four scientific work packages. The first of these aimed to develop the application of high-pressure nuclear magnetic resonance (NMR) measurements for the elucidation of the structure, electronics, and dynamics of enzymes. The second work package was to develop advanced experimental capabilities using time-resolved electron paramagnetic resonance (EPR) (also known as ESR - electron spin resonance) to understand the dynamics of complex biological redox reactions. The objective of the third work package was to exploit time-resolved magnetic field effects (MFEs) to develop a range of new experimental capabilities for the study of short-lived radicals. The final scientific work package aimed to develop new cryo-EPR spectroscopy capabilities and to exploit these approaches to identify very early intermediates and the quantum behaviour of enzyme catalysts. An additional major objective of the MAGIC ITN was the training of 12 ESRs in these scientific areas.
The research carried out for the first work package included the development of expression and purification protocols to provide unlabelled and isotopically labelled pentaerythritol tetranitrate reductase (PETNR), labelled catechol-O-methyl transferase (COMT) and human phosphoglycerate kinase (hPGK). Subsequently backbone and side-chain assignments was completed for these proteins using NMR spectroscopy enabling further NMR spectroscopic investigation to further understand these enzymes and to develop new catalytic models. For the second work package a new flow-flash EPR instrument was designed, optimised and then tested using methylcobalamin, protochlorophyllide-containing systems. Separately, caged molecules were prepared for laser de-caging enzymatic studies and test systems were trialled on a new freeze-quench instrument that was developed in conjunction with MAGIC ITN partner TgK Scientific. Additionally, full-length nitric oxide synthase (nNOS) and labelled calmodulin (CaM) were prepared and significant progress was made by studying these using a variety of EPR techniques. As part of the third work package a novel transient absorption instrumentation was developed to enable investigations of radical pair magneto-sensitivity across all various timescales for chemical and biological systems. For this research the substrate Pchlide, the deuterated cofactor NADPD, the wild type protochorophyllide oxidoreductase (POR), wild type ethanolamine ammonia lyase (EAL) and selected enzyme mutants were produced. A number of enzymatic studies were carried out with these enzymes including enzyme kinetic characterisation and MFE studies are ongoing. For the fourth work package a new EPR spectroscopy cryostat was designed, built and then tested down to below 300 mK (milli-Kelvin) using model systems and a calibrated ruthenium(IV) oxide thermometer. Based on this, future cryo-EPR systems will be designed and optimised to achieve even lower temperatures and to allow light irradiation of the samples. Various chemical and biological systems were studied using low temperature EPR studies and these low temperature EPR spectra were simulated using different software packages. Additional cryo-EPR research was carried out on lanthanide-series complexes, cobalamins and Pchlide.
The main scientific results include the delivery of four new instrument capabilities, access to various biological proteins and a large amount of scientific data and understanding based on the research already carried. out. The instrument capabilities included the development and testing of a cryogenic probe for low temperature (down to less than 1 Kelvin) electron paramagnetic resonance (EPR) measurements. These measurements enable the study and identification of very early intermediate transition states along a reaction coordinate as the extremely low temperatures ‘freeze out’ the nuclear motions and slow reaction paths so that electronic transitions and the quantum behaviour of enzyme catalysts can be studied. Secondly, a continuous flow electron paramagnetic resonance (EPR) apparatus has been made that enables replacement of reactants within the resonator of the EPR instrument allowing accumulation of the signal derived from the reaction paramagnetic species. The data generated provides insight into reaction mechanism research for systems where it is challenging to study short-lived radicals at room temperature because of their transient nature. Thirdly, a new 'flash-smash' sample preparation instrument for time-resolved electron paramagnetic resonance (EPR) has also been build, tested and used with stable 'caged' precursors to generate radicals and reaction intermediates by laser photolysis and trap these at low temperature to enable spectroscopic studies. The fourth instrument capability is a newly developed state-of-the-art variable magnetic field instrumentation for transient absorption measurements that enables magnetic field effect investigations of chemical and biochemical systems on the ultrafast time scales (from femtoseconds to seconds). Various wild-type and mutant enzymes have been prepared, characterised and studied as part of the ITN and these will be used to provide further enzymatic understanding and they have the potential for application in synthetic biology. In addition, wild-type and mutant cryptochromes have been prepared and studied to provide new understanding of their role and they have potential applications in photo-biology and optigonetics. The large amount of scientific data produced as part of the ITN has been used to advance enzymatic and catalytic understanding and more of this will be made available in future publications.
A major component of the MAGIC ITN was the training provided to the 12 ESRs. Initial training focused on general student induction activities, health & safety training, and early scientific training. Subsequently, all the ESRs have had more detailed technical training; attended various lecture courses and symposia; given various presentations on their work; been on initial secondments to the ITN partners; written a literature review for their individual scientific projects; written up a first year report and then passed a first-year viva by independent academics. They have also attended a variety of internal, national and international workshops and conferences; as well as having the opportunity to undertake a range of broader skills training as part of delivering their individual career development plans. Overall, the MAGIC ITN delivered most of the training and the scientific objectives.
A wide range of dissemination activities has taken place in order to communicate to multiple audiences. Throughout the duration of the ITN a dedicated website (www.magic.manchester.ac.uk) has been maintained. This website explains the objectives, details the researchers, highlights the scientific background, and reported on the progress, publications, presentations and other news highlights. Publication in peer-reviewed journals was a key dissemination activity for the network. To date 12 publications have been accepted and a number of these were published in high-impact journals such as “Nature Chemistry”, “Angewandte Chemie” and “Journal of the American Chemical Society”. Another key activity was the presentation of the research at multiple workshops and conferences. To date, over 40 poster presentations and over 50 oral presentations have been given at local, national and international conferences. These included presentations from all 12 of the Early Stage Researchers and from all 4 scientific work packages of the ITN. As part of our outreach activities to the wider scientific community and public, the Early Stage Researchers have been helped run four annual Manchester Institute of Biotechnology Open Days for senior school pupils, been involved in science festivals, participated in over 40 activities/events with school children, and several ‘question and answer’ scientific sessions. Finally, and number of the academics have made YouTube videos explaining their research that are openly available on the internet (https://www.youtube.com/channel/UCXf3CFkyGpa2eIeT5wz_Prw).
The MAGIC award delivered new science and technology capabilities that has and will advance knowledge of the detailed mechanisms of enzyme catalysts. On the longer term, deeper appreciation of enzyme mechanisms through implementation of these innovative science and technology developments will transform understanding of basic catalytic mechanisms. This will aid in the predictive design/redesign of catalysts e.g. for wider applications in the bioeconomy. The award has trained 12 ECRs who are trained to work at the interface of catalysis science and major economic growth areas for the EU including advanced materials, chemicals and biotechnology. MAGIC has therefore contributed to the structuring of an informed workforce and new scientific capabilities that will enable the EU to benefit from opportunities in catalysis science relevant to economic growth and quality of life.