Tools for measuring post-translational modification enzyme activity in vitro and in cells

Project Summary/Abstract My laboratory works at the interface of chemistry and biology on technology development for post-translational modification enzyme activity measurement. We use a broad variety of techniques including peptide chemical biology, spectroscopy, proteomics, cell biology, and analytical chemistry to create tools that help answer biological questions with molecular precision. We have spent the most time on studying and building a toolkit for measuring kinase activity, both in vitro and in live cells. We are currently working on building out our platform technology called KINATEST-ID to characterize kinase preferences, design novel substrates, and apply them in kinase assays with innovative read-outs. Our goals for the next five years are (1) to build up our understanding of kinase-substrate interactions using multiple avenues: in vitro phosphoproteomics experiments to characterize the substrate profiles of many different kinases, comparing substrate preference motifs to try to dissect differences, designing novel artificial substrate tools that could distinguish between the activities of different kinases, and using cutting-edge artificial intelligence-based structure-based computational methods like Rosetta and AlphaFold to characterize and predict determinants of substrate selectivity; and (2) to develop and implement assay read-out detection methods that can meet different levels of technical needs for drug screening, drug target validation, pharmacodynamic monitoring for companion diagnostics, as well as basic study of fundamental questions about enzyme function and interaction with substrates in cells.