Date of Award

August 2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Physics

First Advisor

Ionel Popa

Committee Members

Valerica Raicu, Carol Hirschmugl, David Frick, Jennifer Gutzman

Abstract

Proteins operating under force are involved in several biological processes and perform multiple roles. While the structures and roles of numerous proteins are ubiquitous, their involvement in binding-induced stabilization is currently poorly understood. Most protein systems operating under force interact with their binding partners in a force-dependent manner. Such systems are related to bacterial adhesion, cellular mechano-transduction, and muscle contraction. With a goal of understanding mechanical stability induced through ligand binding, I used single-molecule magnetic tweezers to study several protein systems. This approach involves protein engineering and hetero-covalent attachment chemistry, which, combined with magnetic tweezers, allows us to characterize the unfolding response of single proteins at piconewton forces, over extensive periods, approaching several hours-per-molecule.In this dissertation, I present the findings from three different protein systems focusing on the mechanical stabilization of proteins when interacting with their ligands. First, I explore how bacterial protein L tunes its mechanical stability when binding to its antibody ligand. From the change in mechanical stability of protein L in the presence of antibodies, I determine the binding constant of mechanically reinforced states. I found that the low avidity binding site acts as a mechano-sensor, suggesting a physiological role for this binding interface. Secondly, I delve into talin, a major player in cellular mechano-transduction, and explore how it interacts with a regulatory ligand, Deleted in Liver Cancer-1 (DLC-1), under force. I found the R8 domain of talin is exhibiting folding-unfolding transitions at physiological forces. Interestingly, I also found that the interaction of talin with DLC-1 increases the mechanical stability of R8 domain and prevents its mechanical unfolding. This behavior suggests that the binding of R8 with DLC-1 is stronger than previously thought, thus explaining its role as tumor suppressor. Finally, I investigate how a mutation known to trigger cardiomyopathy in humans affects the mechanical stability of Myosin Binding Protein C (MyBP-C) and alters its interaction with actin. From mechanically unfolding and refolding MyBP-C, I found that the mutation weakens this protein and decreases its folding force. Also, I show that the mutation hampers the binding of MyBP-C through its ligand actin. These two differences between wild type and mutant emphases importance of MyBP-C in regulating the cardiac muscle activity. Overall, this dissertation aims to define the biophysical principles involved in protein-ligand association, which have profound effects on the stability and function of the protein substrate. My results on mechanical response of proteins enhance our understanding on how protein unfolding and refolding in vivo, correlate with ligand binding, might play a gain-of-function role.

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