New approaches for control and visualization of protein conformation in live cells. Public Deposited

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Last Modified
  • March 20, 2019
Creator
  • Stone, Orrin
    • Affiliation: School of Medicine, Department of Pharmacology
Abstract
  • The organization of protein activity in space and time is an essential but poorly understood aspect of cellular signaling. This has begun to change with development of molecular tools that enable visualization or control of protein activity with high spatial and temporal precision. Visualization has been accomplished with small protein domains (affinity reagents) that bind selectively to active proteins, while control has been achieved with light-responsive domains that change a target protein’s localization or active site exposure following irradiation with specific wavelengths of light. However, currently available methods suffer from limitations that have prevented their application to many important biological problems. This dissertation describes two strategies to support broader application of tools for visualization and control of protein activity in live cells. Suitable affinity reagents are not available for many proteins, and even when they are, they generally require extensive optimization and can perturb their target protein’s functionality. In the first study, I detail a new method to generate affinity reagents for large multi-domain proteins by leveraging a high affinity and highly specific protein-peptide interaction. This method provides a streamlined approach to generating absolutely specific affinity reagents with minimal perturbation for a wide range of target proteins. We also demonstrate how our approach can be applied to visualize protein conformation at the single- molecule level. A major challenge in developing light-controlled protein analogs is achieving proper positioning of the attached light-responsive domain to effectively block a target protein’s active site. In the second study, I demonstrate how an engineered protein scaffold can be used to help position light-responsive domains to effectively control target protein activity.
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Rights statement
  • In Copyright
Advisor
  • Jacobson, Kenneth
  • Johnson, Gary
  • Elston, Timothy
  • Kuhlman, Brian
  • Hahn, Klaus
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2018
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