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

核磁共振是一种光谱技术，它能够研究蛋白质在溶液中的结构和动力学，补充了结晶学和电子显微镜的方法。这种动态研究对于了解基本的生化过程、人类健康和疾病很重要，并提供了定向药物发现的途径。然而，尽管核磁共振有用，但它固有的一个问题是大小上限，即较大的蛋白质对研究和产生复杂的光谱变得不敏感，这很难解释。通过同位素标记可以在一定程度上改善这一点，这包括在蛋白质的特定点处选择性地引入具有核磁共振活性核的原子同位素，同时在蛋白质的大多数其他点引入核磁共振无声核。这减少了冗余信号，并提高了灵敏度和光谱分辨率。为了合成这种蛋白质，通常在蛋白质表达的后期阶段将标记有所需原子同位素的原料引入生长介质中。然而，化学合成同位素标记的前体，如L氨基酸或糖，通常使用贵金属催化剂和昂贵的起始原料。此外，选择性有限，同位素纯度降低，从而造成进一步的下游提纯步骤和浪费。由于这些问题，蛋白质核磁共振的应用仍然有限，并为复杂但高度信息丰富的核磁共振技术的普遍使用提供了障碍。提供更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有望满足这一需求，即使用酶进行催化，也称为生物催化或工业生物技术。这可以提供酶反应的好处，如温和的水相反应条件，高的固有选择性，以及可生物降解的催化剂。文森特小组以前的工作已经建立了酶辅助因子重氢方法，该方法可以进一步应用于各种同位素前体的合成的酶促级联反应中。在这一领域的进一步研究可以使核磁共振标记的设计蛋白得到更实际、更常规的使用，并有助于可持续制造、生化发现以及新疗法和药物的开发等领域。该项目属于EPSRC的“制造未来”和“物理科学”主题。因此，该项目的一个目标是开发组合化学和生物催化方法来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)识别可作为特定标记的目标的生化途径，ii)合成用作上述途径的原料的前体，以及iii)使用合成的前体表达和合成用于核磁共振研究的设计标记蛋白质。例如，以前的突破性技术，如‘甲基-TROSY’，使用了特定氨基酸的同位素标记，如异亮氨酸、亮氨酸和缬氨酸，作为弛豫优化的核磁共振的基础。因此，这一目标将有助于核磁共振方法学的发展，并与目标一相结合，为利用核磁共振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.