Periodic Reporting for period 1 - DENOPHECK (Purposing de novo protein scaffolds for the Heck reaction)
Reporting period: 2020-09-01 to 2022-08-31
The overall aim of this fellowship was to develop an enzyme that catalyses the Heck reaction using de novo proteins.
We now live in an age where the consequences of the emissions of greenhouse gases are having observable, detrimental effects on our planet’s ecosystem. Therefore, the need to develop non-toxic, cleaner and lower energy reactions is essential. Palladium(0)-catalysed cross-coupling reactions are a powerful method for generating carbon-carbon and carbon-heteroatom bonds, leading to their extensive use in academia and industry. Among all cross-coupling reactions, it can be argued that the Heck reaction is one of the greenest because the reagents are not prefunctionalised with metals, boron or silicon. Furthermore, it has been demonstrated that the Heck reaction can be performed in water, although truly catalytic examples to date require temperatures up to 140 °C and often more reactive and expensive aryl bromide and aryl iodide reagents.
De novo protein design, the selection of an amino-acid sequence that will fold to a desired protein structure, is a newly established field. Successful designs have furnished a diverse range of protein structures. In contrast to most natural proteins, de novo proteins are well understood, have high thermal stability and are characterised to atomic detail.
Dr Rhys, under the supervision of Prof Höcker & Prof Weber, attempted to purpose a range of natural and de novo proteins to bind artificial co-factors to develop de novo Heckase enzymes. The Covid-19 pandemic was ongoing throughout the two year project. Despite setbacks related to this, and other unforeseeable scientific challenges, we have made significant progress towards the objective of developing an enzyme that can catalyse the Mizoroki-Heck reaction under more environmentally friendly conditions.
We now live in an age where the consequences of the emissions of greenhouse gases are having observable, detrimental effects on our planet’s ecosystem. Therefore, the need to develop non-toxic, cleaner and lower energy reactions is essential. Palladium(0)-catalysed cross-coupling reactions are a powerful method for generating carbon-carbon and carbon-heteroatom bonds, leading to their extensive use in academia and industry. Among all cross-coupling reactions, it can be argued that the Heck reaction is one of the greenest because the reagents are not prefunctionalised with metals, boron or silicon. Furthermore, it has been demonstrated that the Heck reaction can be performed in water, although truly catalytic examples to date require temperatures up to 140 °C and often more reactive and expensive aryl bromide and aryl iodide reagents.
De novo protein design, the selection of an amino-acid sequence that will fold to a desired protein structure, is a newly established field. Successful designs have furnished a diverse range of protein structures. In contrast to most natural proteins, de novo proteins are well understood, have high thermal stability and are characterised to atomic detail.
Dr Rhys, under the supervision of Prof Höcker & Prof Weber, attempted to purpose a range of natural and de novo proteins to bind artificial co-factors to develop de novo Heckase enzymes. The Covid-19 pandemic was ongoing throughout the two year project. Despite setbacks related to this, and other unforeseeable scientific challenges, we have made significant progress towards the objective of developing an enzyme that can catalyse the Mizoroki-Heck reaction under more environmentally friendly conditions.
We have made significant progress in developing an enzyme that catalyses the reaction. We have identified, and experimentally validated, a range of suitable proteins that will make excellent candidates for further testing. We have also established a chemical route to generating new cofactors. Both the protein and cofactor must be combined to make a functional enzyme. Therefore, we have developed a test to measure whether the protein and cofactor combine properly. Lastly, we have identified an enzyme that is catalytically active.
Based on the proof-of-principle work, Dr Rhys presented the work at an international meeting for computational protein designers in September 2022.
Based on the proof-of-principle work, Dr Rhys presented the work at an international meeting for computational protein designers in September 2022.
The research project has laid the foundations for significant progress beyond the state of the art. However, further research will be required to develop the research idea and expand the project's socio-economic impact.
As a direct result of the project, Dr Rhys has achieved his goal of obtaining a Lectureship position at a research-intensive European institute. At Cardiff University, he will apply the leadership and autonomous skills developed during the projects to train the next-generation of students in the UK. He will also use the project results as a foundation to apply for funding to employ more scientists on related projects.
As an indirect result of the project, Dr Rhys along with colleagues Dr Noelia Ferruz and Dr Sergio Romero-Romero raised funding and co-organised a conference, namely Advances in Protein, Folding, Evolution and Design (APFED-22). This was an international conference dedicated to enhancing the European network of scientists whose research is related to proteins. It was a hybrid online/in-person conference conducted over three days, attended by over 200 people, and championed scientists at all career stages with talks and posters from PhD students through to established researchers. The conference speakers were diverse, for example 50% of the speakers were women, and represented research conducted in several European countries, in addition to some intercontinental speakers. APFED-22 has strengthened the European network of researchers and was a fertile ground for newly formed international collaborations.
As a direct result of the project, Dr Rhys has achieved his goal of obtaining a Lectureship position at a research-intensive European institute. At Cardiff University, he will apply the leadership and autonomous skills developed during the projects to train the next-generation of students in the UK. He will also use the project results as a foundation to apply for funding to employ more scientists on related projects.
As an indirect result of the project, Dr Rhys along with colleagues Dr Noelia Ferruz and Dr Sergio Romero-Romero raised funding and co-organised a conference, namely Advances in Protein, Folding, Evolution and Design (APFED-22). This was an international conference dedicated to enhancing the European network of scientists whose research is related to proteins. It was a hybrid online/in-person conference conducted over three days, attended by over 200 people, and championed scientists at all career stages with talks and posters from PhD students through to established researchers. The conference speakers were diverse, for example 50% of the speakers were women, and represented research conducted in several European countries, in addition to some intercontinental speakers. APFED-22 has strengthened the European network of researchers and was a fertile ground for newly formed international collaborations.