Structural and Biochemical Studies of Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII)
CaMKII is a highly conserved, multimeric kinase that represents 1% of your dry brain weight. It plays a key role in mammalian learning and memory. Upon activation, CaMKII holoenzymes can exchange subunits, resulting in a spread of kinase activity. The ultimate goal of this project is to understand CamKII’s subunit exchange at a molecular level. Employed techniques will include: x-ray crystallography, native protein mass spectrometry, fluorescence resonance energy transfer spectroscopy, analytical gel filtration chromatography, molecular cloning, and heterologous protein expression and purification.
High-Throughput Approach to Understanding B and T Cell Kinase Specificity
The tyrosine kinase ZAP-70 plays a crucial role in T cell receptor (TCR) signaling. ZAP-70 is unique among tyrosine kinases, including the paralogous B cell kinase Syk, in that it has very narrow substrate specificity. Although this specificity is required for stringent control of TCR signaling, it is not clear how ZAP-70 recognizes its substrates. To understand the structural basis of this specificity, we will use chemically synthesized peptide-ATP conjugates to obtain co-crystals and solve high-resolution complex structures. In addition, we have developed high-throughput methods to simultaneously determine the activity of ZAP-70 towards thousands of substrate mutants.
Investigation of the RING-Type E3 Ligase Cbl From Multiple Organisms
This project involves understanding the relationship between tyrosine kinases and Cbl – an E3 ligase that ubiquitylates these kinase substrates, targeting them for degradation. Specifically, we are interested in the protein-protein interactions of tyrosine kinases and the tyrosine kinase-binding domain of Cbl homologues from distantly related organisms (e.g., D. discoideum, S. rosetta, and C. elegans). In this project, we will use in vitro biochemical assays, enzyme kinetics, and x-ray crystallography.