Biocatalytic Synthesis of Selectively Isotopically Labelled Biomolecules for Preparation of Labelled Proteins & NMR Structure Determination

Nuclear magnetic resonance (NMR) is a spectroscopic technique which enables study of the structure and dynamics of proteins in solution, complementing crystallographic and electron
microscopy approaches. Such dynamical studies are important in understanding fundamental biochemical processes, human health and disease, and offer routes towards targeted drug
discovery. However, despite its usefulness, an inherent issue with NMR is the upper size limit, whereby larger proteins become insensitive to study and produce complicated spectra, which are difficult to interpret. This can be somewhat improved through isotopic labelling, which involves selectively introducing atomic isotopes with NMR-active nuclei at specific points of the protein, while introducing NMR-silent nuclei at most other points of the protein. This reduces redundant signals, and improves sensitivity and spectral resolution. To synthesise such proteins, feedstocks labelled with the required atomic isotopes are typically introduced into the growth media at late stages of protein expression. However, chemical synthesis of the isotopically labelled precursors, such as L-amino acids or sugars, often uses precious-metal catalysts and expensive starting materials. Furthermore, there is limited selectivity and lowered isotopic purity, which creates further downstream purification steps and waste. Due to these issues, applications for protein NMR remain limited and present a barrier for common use of complex, yet highly information-rich NMR techniques.

Synthetic methods offering greener chemistry are in demand as ever, and a rapidly developing field poised to meet this need is using enzymes for catalysis, otherwise known as biocatalysis or industrial biotechnology. This can offer the benefits of enzymatic reactions such as mild, aqueous reaction conditions, high inherent selectivities, and biodegradable catalysts. Previous work in the Vincent group has established enzyme cofactor deuteration methods, which can be further applied in enzymatic cascades for the synthesis of various isotopic precursors. Further research in this area could enable more practical, routine use of NMR-labelled designer proteins, and contribute towards areas encompassing sustainable manufacturing, biochemical discoveries, and development of novel therapeutics and pharmaceuticals.

This project falls within the "manufacturing the future" and "physical sciences" EPSRC themes. Therefore, one aim of this project is to develop combinatorial chemo- and bio-catalytic
approaches towards synthesising isotopically labelled precursors such as amino acids, sugars, and their various derivatives. This will be completed through the following objectives:
i) identification of biochemical pathways that can be targeted for specific labelling, ii) synthesis of precursors to use as feedstock into the aforementioned pathways, and iii) expression and synthesis of designer labelled proteins for NMR study using the synthesised precursors.

A second aim of this proposal is to explore how selective isotopic labelling can be exploited for specialised protein NMR. For example, previous breakthrough techniques such as 'methyl-
TROSY' have used isotopic labelling at specific amino acids such as isoleucine, leucine, and valine as the foundation of relaxation-optimised NMR. Therefore, this aim will contribute to
NMR methodology development and in combination with aim one, offers further routes to studying proteins by NMR.

The primary impact of the OxICFM CDT will be the highly-trained world-class scientists that it delivers. This impact will encompass both the short term (during their doctoral studies), the medium term (subsequent employment) and ultimately the longer timescale defined by their future careers and consequent impact on science, engineering and policy in the UK.

The impact of OxICFM students during their doctoral studies will be measured by the culture change in graduate training that the Centre brings about - in working at the interface between inorganic synthesis and manufacturing, and fostering cross-sector industry/academia working practices. By embedding not only from larger companies, but also SMEs, we have developed a training regime that has broader relevance across the sector, and the potential for building bridges by fostering new collaborations spanning enormous diversity in scientific focus and scale. Moreover, at a broader level, OxICFM offers to play a unique role as a major focus (and advocate) for manufacturing engagement with academic inorganic synthetic science in the UK.

From a scientific perspective, OxICFM will be uniquely able to offer a broad training programme incorporating innovative and challenging collaborative projects spanning all aspects of fundamental and applied inorganic synthesis, both molecular and materials based (40+ faculty). These will address key challenges in areas such as energy provision/storage, catalysis, and resource provision/renewal necessary to enhance the capability and durability of UK plc in the medium term. To give some idea of perspective, the output from previous CDTs in Oxford's MPLS Division include two start-up companies and in excess of 30 patents.

It is not only in the industrial and scientific realms that students will have impact during their timeframe of their doctorate. Part of the training programme will be in public engagement: team-based challenges in resource development/training and outreach exercises/implementation will form part of the annual summer school. These in turn will constitute a key part of the impact derived from the CDT by its engagement with the public - both face-to-face and through electronic/web-based media. As the centre matures, our aspiration is that our students - from diverse backgrounds - will act as ambassadors for the programme and promote even higher levels of inclusion from all parts of society.

For our partners, and businesses both large and small in the manufacturing sector, it will be our students who are considered the ultimate output of the OxICFM CDT. Our programme has been shaped by the need of such companies (frequently expressed in preliminary discussions) to recruit doctoral graduates who can apply themselves to a broad spectrum of multi-disciplinary challenges in manufacturing-related synthesis. OxICFM's cohort-based training programme integrates significant industry-led training components and has been designed to deliver a much broader skill set than standard PhD schemes. The current lack of CDT training at the interface of inorganic chemistry and manufacturing (and the relevance of inorganic molecules/materials to numerous industrial sectors) heightens the need for - and the potential impact of - the OxICFM CDT. Our students will represent a tangible and valuable asset to meet the long-term skills demand for scientists to develop new materials and nanotechnology identified in the UK Government's 2013 Foresight report.

In the longer term, the broad and relevant training delivered by OxICFM, and the uniquely wide perspective of the manufacturing sector it will deliver, will allow our graduates to obtain (and thrive in) positions of significant responsibility in industry and in research facilities/institutes. Ultimately we believe that many will go on to be future research leaders, driving innovation and changing research culture, and thereby making a lasting contribution to the UK economy.