Conformational Disorder in Protein Function and Pathogenic Aggregation

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The tools of structural biology (i.e., X-ray crystallography and structural NMR) provide exquisitely detailed 'snapshots' of the 'native structure' of proteins. However, if all proteins were as static as they appear in these 'snapshots', most would be completely non-functional. To achieve biological activity, proteins must access specific, higher energy conformations within an ensemble of 'native-like' structures that are populated via thermally-driven fluctuations known as conformational dynamics. The objective of my group's research is to understand specifically how conformational dynamics drive protein function and, in some cases, the adoption of pathogenic structures that 'clump together' to form dangerous aggregates called amyloids. To do this, we need to know what these higher energy structures look like, but identifying them is no easy task because they tend to be short-lived, weakly populated at equilibrium and very similar to the 'ground-state' structure that dominates the native ensemble.**In work funded by our previous (first) NSERC Discovery grant, my group introduced a set of mass spectrometry-coupled microfluidic chips that enable characterization of 'higher energy' protein conformations using a technique called Hydrogen/Deuterium Exchange. In the present research program, we will use these devices to learn about the dynamic processes that underlie protein function, specifically how they enable catalysis, allostery (action at a distance) and pathogenic aggregation. In the case of catalysis, for instance, our aim is to understand how (or if) conformational dynamics guide the enzyme along it's catalytic reaction pathway. To do this, we will characterize dynamics in a 'normal' enzymatic reaction and one that has been 'slowed' by substituting a heavy isotope at a critical atom on the substrate (resulting in a 'primary kinetic isotope effect'). If the dynamics are unaffected by the change in reaction rate, this would indicate that dynamics are not directly linked to catalysis (or at least not to the rate-limiting step in the catalytic mechanism). In the case of allostery, we are interested in learning how subtle changes in structure or dynamics can 'transmit' information from a binding or covalent modification site to distant parts of the protein. Understanding this is crucial for being able to predict how binding, modification or mutation at peripheral sites will influence protein function. In the case of pathogenic aggregation, we are interested in understanding changes in the conformational ensemble that cause weakly structured proteins (or intrinsically disordered proteins, which have essentially no set structure) to be come amyloidogenic. The answer must lie in the regions of 'residual structure' that persist even in proteins that are largely disordered. Our rapid H/D exchange labeling techniques give us a unique capability to characterize residual structure in intrinsically disordered proteins (and disordered regions of proteins), which will allow us to investigate how binding or covalent modification-driven shifts in the 'disordered' ensemble modulate binding specificity and biological activity. **The proposed research is aimed squarely at advancing our basic knowledge of protein function. However, like most biologically-linked research in the natural sciences, our hope is that this knowledge will ultimately provide new avenues for the treatment of disease. Our insights are of particular relevance to conformational pathogenesis, and to the breakdown of protein interaction networks in cancer and neurodegenerative disease.