Visualising Glycoprotein Interaction Dynamics

在我们的细胞中，对生命至关重要的功能——从抵抗感染到复制dna——都是由蛋白质执行的。这些是由一系列复杂的机制调控的，包括蛋白质相互作用和化学修饰。一种常见的蛋白质修饰是糖基化，这是一种复杂的、非模板驱动的过程，它将复杂的碳水化合物分子或聚糖添加到单个氨基酸中。据估计，人类一半的蛋白质是糖基化的，而糖基结构对蛋白质有巨大的影响。通过与其他蛋白质和生物分子的相互作用，与蛋白质结合的聚糖可以改变蛋白质的结构和功能，改变蛋白质在细胞中的去向及其稳态。糖基化的重要性可以通过一系列被称为先天性糖基化疾病的人类疾病来说明，在这些疾病中，糖基化生物合成的轻微缺陷会导致严重的多系统功能障碍、器官衰竭甚至过早死亡。糖基化与生物制药行业密切相关。它影响到单克隆抗体和其他治疗性“生物制剂”的安全性和有效性，这是一类快速增长的用于治疗包括癌症和自身免疫性疾病在内的疾病的药物。聚糖对于包括流感和埃博拉在内的许多病毒也是至关重要的，这些病毒通过隐藏在动态、密集的“聚糖屏蔽”下来逃避我们的免疫系统。尽管糖基化的重要性显而易见，但令人惊讶的是，我们对聚糖如何影响它们所结合的蛋白质的性质和相互作用知之甚少。缺乏知识的一个主要原因是聚糖很难研究。它们形成了一组令人眼花缭乱的复杂动态结构，即使是当今最强大的工具也无法对它们进行分析。由于糖蛋白在生物分子相互作用中的普遍存在，为了理解蛋白质的功能，揭示其固有的结构复杂性是至关重要的，但需要创造性和开创性的方法。我计划通过开发一种以新方式结合现有技术的方法来解决目前工具的严重缺乏，创造一种强大的方法来捕获聚糖和其他分子之间发生的相互作用。我们的方法将结合化学交联，将化学“标签”结合到聚糖中，使交联成为可能。化学交联可以监测甚至是短暂的蛋白质相互作用或动态特性，但目前不适合聚糖和代谢糖蛋白工程。我们还将使用尖端的计算和单分子质量测量技术来收集补充数据，以帮助我们解释交联方法的信息。作为这项工作的一部分，我们将应用我们的新方法来研究流感，埃博拉糖基化，人类抗体受体识别和单克隆抗体的糖基化，从而扩大我们对糖基化在健康，疾病和药物开发中的功能的了解。我的未来领袖奖学金的目的是改变我们研究和可视化糖蛋白的能力。我相信我们可以创造出我们需要的工具来解决涉及糖蛋白的复杂的长期生物学问题，我计划开发这样一个工具。我的方法将通过提供糖蛋白相互作用动力学的前所未有的详细水平，使新的生物学发现成为可能。除了预计将直接从该项目中产生的重要发现外，该方法还代表了糖蛋白研究的范式转变。

In our cells, the functions that are vital for life-from fighting infection to replicating DNA-are carried out by proteins. These are regulated by a complex array of mechanisms, including protein-protein interactions and chemical modifications. A common protein modification is glycosylation-an intricate, non-template driven process that adds complex carbohydrate molecules, or glycans, to individual amino acids. It is estimated that half of human proteins are glycosylated, and glycan structures have a huge impact on the protein. By interacting with other proteins and biomolecules, glycans bound to a protein can alter that protein's structure and function, where it is goes in a cell, and its homeostasis. The importance of glycoslation is illustrated by a set of human conditions known as the Congenital Disorders of Glycosylation, where mild defects in glycan biosynthesis lead to severe multisystem malfunction, organ failure and even premature death. Glycosylation is highly relevant to the biopharmaceutical industry. It affects the safety and efficacy of monoclonal antibodies and other therapeutic 'biologics'-a rapidly growing class of drugs for treating conditions including cancers and autoimmune diseases. Glycans are also of central importance to many viruses, including influenza and Ebola, which evade our immune systems by hiding under a dynamic, dense 'glycan shield'. Despite the clear importance of glycosylation, we know surprisingly little about how glycans influence the properties and interactions of the proteins they are bound to. One of the main reasons for this lack of knowledge is simply that glycans are very difficult to study. They form a bewildering array of complex, dynamic structures, and their analysis eludes even today's most powerful tools. Due to the prevalence of glycoproteins in biomolecular interactions, unravelling their inherent structural complexity in order to understand protein function is fundamentally important but requires creative and pioneering methodologies. I plan to address the current critical lack of tools by developing an approach that combines existing techniques in a new way, creating a powerful method for capturing the interactions that take place between glycans and other molecules. Our approach will combine chemical crosslinking-which makes it possible to monitor even short-lived protein interactions or dynamical properties but is currently unsuited for glycans-and metabolic glycoprotein engineering-to incorporate chemical 'tags' into glycans that will enable crosslinking. We will also use cutting-edge computational and single-molecule mass measurement techniques to gather complementary data to help us interpret the information from the crosslinking approach. As part of this work, we will apply our new approach to study glycosylation in influenza, Ebola glycosylation, human antibody-receptor recognition and monoclonal antibodies, thus expanding our knowledge of glycan function in health, disease and drug development. The aim of my Future Leaders Fellowship is to transform our ability to study and visualise glycoproteins. I believe that we can create the tools we need to address complex long-standing biological questions involving glycoproteins, and I plan to develop such a tool. My approach will enable new biological discoveries by providing an unprecedented level of detail about glycoprotein interaction dynamics. In addition to the important discoveries anticipated to arise directly from this project, the method represents a paradigm shift for the study of glycoproteins.