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

选择性同位素标记生物分子的生物催化合成用于制备标记蛋白

• 批准号: 2581241
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2581241
• 关键词: Biocatalytic; Synthesis; Selectively; Isotopically; Labelled

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.

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，是对晶体学和电子显微镜方法的补充。这种动力学研究对于理解基本生化过程、人类健康和疾病非常重要，并为靶向药物的发现提供了途径。然而，尽管核磁共振很有用，但其固有的问题是尺寸上限，较大的蛋白质对研究变得不敏感，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。为了合成此类蛋白质，通常在蛋白质表达的后期将用所需原子同位素标记的原料引入生长培养基中。然而，同位素标记前体（例如 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，选择性有限且同位素纯度降低，这会产生进一步的下游纯化步骤和浪费。由于这些问题，蛋白质 NMR 的应用仍然受到限制，并且对复杂但信息丰富的 NMR 技术的普遍使用构成了障碍。人们对提供更绿色化学的合成方法的需求一如既往，而为满足这一需求而快速发展的领域是使用酶进行催化，也称为生物催化或工业生物技术。这可以提供酶促反应的优点，例如温和的水性反应条件、高固有选择性和可生物降解的催化剂。 Vincent小组之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合成各种同位素前体的酶级联中。该领域的进一步研究可以使核磁共振标记的设计蛋白更加实用、常规使用，并为可持续制造、生化发现以及新型疗法和药物开发等领域做出贡献。该项目属于 EPSRC 的“制造未来”和“物理科学”主题。因此，该项目的目标之一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，例如氨基酸、糖及其各种衍生物。这将通过以下目标来完成：i) 鉴定可针对特定标记的生化途径，ii) 合成前体作为上述途径的原料，以及 iii) 使用合成的前体表达和合成设计者标记的蛋白质，用于 NMR 研究。该提案的第二个目标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，之前的突破性技术（例如“甲基-TROSY”）已使用特定氨基酸（例如异亮氨酸、亮氨酸和缬氨酸）的同位素标记作为弛豫优化 NMR 的基础。因此，这一目标将有助于核磁共振方法学的发展，并与目标一相结合，为通过核磁共振研究蛋白质提供进一步的途径。