Affiliation: School of Medicine, Department of Biochemistry and Biophysics
Cell behavior results from the precise orchestration of molecular activity in time and space. The need to understand dynamics of proteins in the context of living systems has recently led to the development of a remarkable suite of protein ‘switches’, engineered domains and other approaches that cause proteins to respond to small molecules or light, enabling us to control the spatiotemporal dynamics of protein-protein interactions, posttranslational modifications, conformational change, and subcellular localization. However, existing methods suffer from many disadvantages including increased basal activity before protein activation, slow kinetics, difficulty in delivery and expression, and inefficient activation. This dissertation describes two strategies to manipulate protein activity to interrogate the role of the protein of interest in cell motility. In the first study, I developed a ligand-controlled switch to manipulate activities of various kinases dynamically. In the second study, I developed a novel and generalizable approach to control protein activity by splitting target proteins and regulating their reassembly using a ligand or light. Both methods were used to investigate the dynamics of proteins including kinases and guanine nucleotide exchange factors in cell motility.