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

核磁共振（NMR）是一种光谱技术，可以研究溶液中蛋白质的结构和动力学，补充晶体学和电子显微镜方法。这种动力学研究对于理解基本的生物化学过程、人类健康和疾病非常重要，并为靶向药物发现提供了途径。然而，尽管其有用，但NMR的固有问题是尺寸上限，由此较大的蛋白质变得对研究不敏感并产生难以解释的复杂光谱。这可以通过同位素标记得到一定程度的改善，同位素标记涉及在蛋白质的特定点选择性地引入具有NMR活性核的原子同位素，同时在蛋白质的大多数其他点引入NMR沉默核。这减少了冗余信号，并提高了灵敏度和光谱分辨率。为了合成这样的蛋白质，通常在蛋白质表达的后期阶段将用所需原子同位素标记的原料引入生长培养基中。然而，同位素标记的前体（如L-氨基酸或糖）的化学合成通常使用贵金属催化剂和昂贵的起始材料。此外，存在有限的选择性和降低的同位素纯度，这产生进一步的下游纯化步骤和废物。由于这些问题，蛋白质核磁共振的应用仍然有限，并且为复杂但信息丰富的核磁共振技术的普遍使用带来了障碍。提供绿色化学的合成方法一如既往地受到需求，而一个快速发展的领域准备满足这一需求，即使用酶进行催化，也称为生物催化或工业生物技术。这可以提供酶促反应的益处，例如温和的水性反应条件、高的固有选择性和可生物降解的催化剂。Vincent小组以前的工作已经建立了酶辅因子氘化方法，该方法可以进一步应用于各种同位素前体的酶级联合成。在这一领域的进一步研究可以使NMR标记的设计师蛋白质的更实用，更常规的使用，并有助于包括可持续制造，生物化学发现和新疗法和药物开发的领域。该项目属于“制造未来”和“物理科学”EPSRC主题的福尔斯。因此，该项目的一个目的是开发组合化学和生物催化方法，以合成同位素标记的前体，如氨基酸，糖及其各种衍生物。这将通过以下目标来完成：i）鉴定可以针对特定标记的生物化学途径，ii）合成前体以用作上述途径的原料，以及iii）使用合成的标记物表达和合成用于NMR研究的设计师标记的蛋白质。该提议的第二个目的是探索如何利用选择性同位素标记用于专门的蛋白质NMR。例如，以前的突破性技术，如“甲基-TROSY”，已经在特定的氨基酸，如异亮氨酸，亮氨酸和缬氨酸上使用同位素标记作为弛豫优化NMR的基础。因此，这一目标将有助于NMR方法学的发展，并与目标一相结合，为NMR研究蛋白质提供了进一步的途径。

Nuclear magnetic resonance (NMR) is a spectroscopic technique which enables study of the structure and dynamics of proteins in solution, complementing crystallographic and electronmicroscopy approaches. Such dynamical studies are important in understanding fundamental biochemical processes, human health and disease, and offer routes towards targeted drugdiscovery. 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-catalyticapproaches 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 toNMR methodology development and in combination with aim one, offers further routes to studying proteins by NMR.