Design and application of peptide-based fluorescent biosensors for protein tyrosine kinases
Protein tyrosine kinases are essential components of cell signaling pathways, as well as highly important drug targets for various types of cancer(1, 2). Tyrosine kinases catalyze the phosphorylation of tyrosine residues of certain proteins. Tyrosine phosphorylation can lead to changes in enzyme activity, protein conformation, or protein-protein interactions, and deregulation of these processes is known to be responsible for a variety of mechanisms that eventually lead to oncogenesis. Tyrosine kinases are now not only major targets of drug discovery efforts, but also the key for answering emerging critical questions in cancer biology(3, 4). Therefore, monitoring the dynamics of tyrosine kinase activity has significant value from fundamental research to various application, and the development of corresponding tool could provide deeper insights into the biological questions that need to be answered. Existing strategies for kinase activity detection usually use radioactive materials, antibodies, or proteomic techniques to monitor native substrates(5, 6), but such strategies also have limitations that affect their ability to report dynamic endogenous kinase activities. In this work, we applied novel strategies to develop peptide-based biosensors to detect kinase activity. We first investigated different methods for in silico design of peptide biosensors with tunable specificity and other chemical properties, and we examined this workflow by experimental validation of both specific kinase substrates and universal kinase substrates. We also investigated methods to integrate lanthanide binding capabilities into existing kinase substrates. Using this information, we then developed kinase biosensors for high-throughput screening compatible, antibody-free time-resolved fluorescence/FRET kinase assays. The biosensors developed by these workflows were also modified with cell-penetrating peptides and small molecule fluorophores for the purpose of fluorescence lifetime imaging, and the feasibility of using biosensors designed for Src and Jak2 in fluorescence lifetime imaging was investigated. In summary, we demonstrated the design of peptide-based biosensors and a variety of their potential applications.
Parker, Purdue University.
Analytical chemistry|Pharmacy sciences
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