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

Lead Research Organisation: University of Oxford
Department Name: OxICFM CDT

Abstract

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.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/S023828/1 31/03/2019 29/09/2027
2581241 Studentship EP/S023828/1 30/09/2021 29/09/2025 Alison Tam