Visualising Glycoprotein Interaction Dynamics

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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.