Exploring the Conformational Landscape and Stability of Aurora A Using Ion-Mobility Mass Spectrometry and Molecular Modeling.

Protein kinase inhibitors are highly effective in treating diseases driven by aberrant kinase signaling and as chemical tools to help dissect the cellular roles of kinase signaling complexes. Evaluating the effects of binding of small molecule inhibitors on kinase conformational dynamics can assist in understanding both inhibition and resistance mechanisms. Using gas-phase ion-mobility mass spectrometry (IM-MS), we characterize changes in the conformational landscape and stability of the protein kinase Aurora A (Aur A) driven by binding of the physiological activator TPX2 or small molecule inhibition.

Ion Mobility-Mass Spectrometry to Evaluate the Effects of Protein Modification or Small Molecule Binding on Protein Dynamics.

Ion mobility-mass spectrometry (IM-MS) of intact protein complexes under native conditions is a powerful tool for the analysis of protein complexes and protein-ligand interactions, permitting insight into ligand-induced changes in protein conformation. Here we describe a procedure for analyzing the effects of phosphorylation and/or inhibitor binding on protein kinase conformational flexibility using Protein Kinase A (PKA) as a model system. By calculating the protein collision cross section (CCS) before and after inhibitor binding, and additionally by performing collision-induced unfolding (CIU), we can establish the effects of protein modification or small molecule binding on protein dynamics.

Determination of Phosphohistidine Stoichiometry in Histidine Kinases by Intact Mass Spectrometry.

Protein histidine phosphorylation has largely remained unexplored due to the challenges of analyzing relatively unstable phosphohistidine-containing proteins. We describe a procedure for determining the stoichiometry of histidine phosphorylation on the human histidine kinases NME1 and NME2 by intact mass spectrometry under conditions that retain this acid-labile protein modification. By characterizing these two model histidine protein kinases in the absence and presence of a suitable phosphate donor, the stoichiometry of histidine phosphorylation can be determined.