1. PIKK kinases and kinase complexes
Protein kinases are amongst the largest family of enzymes and represent about 2% of all genes encoded by the human genome. Protein kinases regulate almost all cellular pathways, and it is estimated that up to one third of all cellular proteins are phosphorylated by kinases. The signaling pathways established by kinases form interconnected networks, which allow a cell to respond rapidly to changes to its extracellular and internal environments. Mutations or dysregulation of kinases are associated with diseases including cancer, diabetes, and neurodegenerative disorders. We are particularly interested in the phosphoinositide-3 kinase-related kinase (PIKK) family of kinases. Members of family of kinases are distinguished by their extraordinarily large size and a propensity to form even larger multi-protein assemblies. PIKK members play central roles in regulating cell growth and proliferation, responses to DNA damage and RNA splicing errors, and transcription activation. We use single particle EM, yeast genetics and other biochemical and structural approaches to characterize the molecular structure, function, and regulation of these kinases and their complexes.
2. Single particle electron microscopy (EM)
Single particle EM has emerged as an indispensible tool to characterize the structures of large proteins and multi-protein assemblies, which are often difficult to be isolated in huge quantities due to low abundance and inherent instability. This structural approach involves imaging individual “particles” from stained or frozen specimens (cryo-EM) with a transmission electron microscope (TEM), and reconstructing the three-dimensional (3D) structures from these two dimensional (2D) images with specialized image processing algorithms. The electron density map obtained by EM could be used to fit the high resolution X-ray or NMR structures of individual components to generate a pseudoatomic model of the entire complex. Because it does not involve crystallization, single particle EM has the capability to visualize different conformational states of dynamic proteins and protein complexes. We are interested in promoting the application of this technology in other research fields as well as developing new methods and procedures to facilitate the characterization of more heterogeneous and inherently dynamic proteins and macromolecular assemblies.
