Characterization of Structures and Dynamics of Intrinsically Disordered Domain and Proteins Using Time Resolved-Hydrogen Deuterium Exchange Mass Spectrometry

Proteins are inherently dynamic. Virtually all of the processes that underlie biological activity, including binding partner recognition, catalysis and allostery among many others, require transient adoption of specific high energy conformations. While many proteins contain highly dynamic regions or Intrinsically Disordered Domains (IDDs), Intrinsically Disordered Proteins (IDPs) are free of well-defined structural features altogether. These domains/proteins play integral roles in a wide variety of biological processes, but are often associated with neurodegenerative disease and cancer. Hence it is crucial to study these biomacromolecules to understand the structural underpinnings of their biological functions as well as the factors that drive disease pathology. However, many of the current high resolution structural techniques (like X-ray crystallography and NMR) are not feasible for such studies because of their reliance on the presence of a well-defined native conformation. An emerging structure labeling technique called Time Resolved-Hydrogen Deuterium Exchange (TR-HDX) can offer an alternative strategy. In this approach, structure-dependent hydrogen/deuterium labelling is measured on the millisecond timescale, providing an exquisitely sensitive picture of the weak hydrogen bonding networks that impart residual structure in IDDs and IDPs. In this work, TR-HDX was implemented onto microfluidic chip to achieve solution phase site-specific analysis of the structures and dynamics of p53 N-terminal transactivation domain and tau protein. Gas-phase TR-HDX was also implemented to study gas-phase protein structures that become populated during Differential Mobility Spectrometry (DMS) ion mobility analyses. Critical advances include the discovery and characterization of an amyloidogenic tau intermediate that may underlie Alzheimers disease pathology, a dynamics-based model for recruitment of co-factors to cancer protein p53 upon phosphorylation, a detailed account of how a set of anti-amyloid drug candidates affect tau structure and dynamics, and development of a method to predict solution-phase protein stability from DMS-HDX measurements.