Our research focuses on describing the effects of reversible, covalent phosphorylation (His-Asp two component signalling) and noncovalent phosphates (cyclic nucleotide monophosphate (cAMP), ATP, ADP and AMP) on protein conformational dynamics. Ongoing research areas include role of phosphodiesterases (PDEs) in cAMP homeostasis and allosteric effects of histidine phosphorylation and phosphotransfer to aspartates in two-component signalling.
How viruses use their quaternary coat protein structures as thermodynamic sensors for detecting entry into a conducive host cell environment is largely unknown. Amide Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) can be used to monitor whole viral particle dynamics in solution. A map of the quaternary structural changes in disassembly state offers critical insights into cryptic epitopes as novel vaccine targets. Our research has pioneered structural mass spectrometry characterization of temperature and divalent cation-dependent conformational changes on Dengue virus and Turnip Crinkle Virus (TCV) and mapped the specific disassembly processes in these two model virus systems. We have extended these studies to deconstruct the whole viral particle-antibody (DENV2-2D22) complex. These results provide powerful new targets for therapeutic antibody design as well as offer fundamental insights into strategically timed viral disassembly processes which can be extended to all viruses.
We seek to probe the dynamics of membrane proteins with an emphasis on signalling receptors with transmembrane domains by a combination of biophysical methods including cross-linking, fluorescence and structural mass spectrometry. Ongoing projects include EnvZ osmosensing receptor, OmpF porin in E. coli and GPCRs in eukaryotes. The research focus is on describing lipid-mediated changes in receptor dynamics and transmembrane signalling.
HDX-MS is being applied to identify lead fragment compounds by mapping the regions of the protein that show altered dynamics in the presence of weak affinity ligands. These maps are being used to rank and develop weak affinity fragments into more specific high affinity leads.
A powerful method to probe dynamics in solution is amide hydrogen/deuterium exchange (HDX) which uses polypeptide backbone amide hydrogens as conformational probes. This exchange is dependent on structure (H-bonding), neighborhood effects and macromolecular interactions and is a readout of conformational flexibility in the millisecond-minute timescale. HDX-MS is especially useful as a comparative technique for probing conformational dynamics of proteins under biologically-relevant perturbations including post-translational modifications, protein, lipid, nucleic acid and ligand interactions. Our group seeks to develop HDXMS and ion mobility mass spectrometry methods and applications in protein dynamics.
Molecular dynamics (MD) simulations are used to model biophysical information from HDX-MS to develop models for allostery in signalling proteins, mapping protein-protein interactions and resolving timescales of protein dynamic processes.