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Monitoring Cell Signaling Events and Supramolecular Protein Assembly with Peptide-Based Fluorescent Probes

Garrett R Casey, University of Nebraska - Lincoln


The maintenance of a robust cell signaling network is critical to numerous cellular processes including growth, differentiation, migration, division, and apoptosis. A common theme in cellular signaling is the oligomerization of transmembrane proteins leading to the activation of protein phosphatases (PPs) and/or protein kinases (PKs) inside the cell. These later enzymes then rapidly alter the phosphorylation state of target substrates and enables cells to quickly respond to their external environment. Therefore, chemical tools directly monitoring the enzymatic activity of PPs and PKs can provide insight into changes in cellular signaling dynamics associated with disease. Moreover, the ability to control protein oligomerization, or supramolecular assembly, could provide a means to validate the roles of PP and PK activity in disease. This dissertation focuses on the development and application of peptide-based fluorescent probes to monitor the activity of PPs and PKs as well as induce supramolecular assembly of membrane proteins on living cells. This dissertation begins with an overview of existing tools to monitor PP activity (Chapter 1). Next, detailed protocols are presented for the synthesis and application of a new class of PP activity probes (Chapter 2). This approach is then used to generate a selective probe for a PP involved in tumor biology (Chapter 3). Building off of this work, a high-throughput assay was developed to monitor the activity of a critical PK involved in cell growth signaling (Chapter 4). Lastly, in a separate but related work, design principles for the evolution of miniprotein hosts capable of supramolecular assembly with a fluorogenic guest are disclosed (Chapter 5). Taken together, this dissertation provides a set of tools to monitor PP and PK activity as well as the control of protein oligomerization. It is envisioned that these tools could be used to dissect signaling perturbations associated with human disease within future studies.

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Recommended Citation

Casey, Garrett R, "Monitoring Cell Signaling Events and Supramolecular Protein Assembly with Peptide-Based Fluorescent Probes" (2019). ETD collection for University of Nebraska-Lincoln. AAI22583429.