Author
Bienick, Matthew S.Issue Date
2019Advisor
Ghosh, Indraneel
Metadata
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The University of Arizona.Rights
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author.Embargo
Release after 01/10/2022Abstract
Selectively modulating the activity of a desired enzyme in cells is a major goal in protein design and can aid in the development of methods for understanding and rewiring cell-signaling pathways. Traditional approaches such as small molecule inhibitors may be difficult to design for selectively targeting a particular enzyme, while gene knockdown methods often lead to compensatory cellular mechanisms thus obscuring true cell signaling. To overcome these issues, several methods have been developed for the specific control of protein activity for both understanding cell signaling as well as generating a portfolio of tunable proteins for a variety of applications. Herein, we demonstrate the design and validation of a potentially general allosteric approach for gating protein kinase and protein phosphatase activity. We have utilized the Bcl‐2 protein family and their small molecule inhibitors to design a system where specific BH3 domains are inserted into a kinase at predetermined, non-homologous positions. BH3 domains are unstructured but adopt an α-helical conformation upon interaction with a protein binding partner, such as Bcl-xL. Thus, in our system Bcl-xL acts as a poison and allosterically inhibits the function of BH3-inserted-enzymes. Subsequently, the addition of a small molecule inhibitor, ABT-737 or A-155463, binds to and displaces Bcl-xL, acting as an antidote, thus restoring enzymatic activity. We show that this method allows for controlling the activity of several protein tyrosine kinases and phosphatases with a small molecule in a dose-dependent fashion both in vitro and in mammalian cells. While our first-generation approach for engineering allostery within enzymes was successful, the protein-protein interactions (PPI) used for controlling the enzymes, Bcl-xL and Bad, are both known to interact with other endogenous proteins. In order to develop a biologically silent PPI we utilized a previously identified selective mutant of Bcl-xL, Bcl-xL R139A, and employed phage display to select for a mutant Bad peptide, MB_IW2, that specifically interacts with Bcl-xL R139A. This selected PPI successfully displayed selectivity in the context of our allosterically regulated kinases in mammalian cells over other known wild type protein binding partners. This design and evolution approach further expands the toolkit for selective enzyme control. In another project, we used sequence alignment methods coupled with structure-guided mutagenesis to design a new split-luciferase for monitoring PPIs. Simple strategies to monitor PPIs in cells allows for biophysical characterization of the proteome in its native environment while providing convenient assays for developing inhibitors for PPIs. In this study we describe the design and characterization of a new split-firefly luciferase (split-Fluc) that can be used for measuring PPIs. We show that our sequence dissimilarity (SD) based design results in several new first-generation split-Fluc with signal to background of > 20-fold. Importantly, second-generation split-Fluc PPI sensors designed through structure-guided mutagenesis of the fragmented enzyme interface resulted in multiple sensors with signal to background of > 200. We demonstrate that these new split-Fluc sensors can be readily used to monitor PPIs and their inhibition in mammalian cells. Taken together, our efforts have expanded the toolbox for exerting specific user-defined control over protein function and for monitoring protein interactions. These new tools will potentially contribute to the understanding of cell signaling mechanisms and in the development of new therapeutics.Type
textElectronic Dissertation
Degree Name
Ph.D.Degree Level
doctoralDegree Program
Graduate CollegeBiochemistry