Undergraduate Honors Thesis

 

A computationally designed protein-ligand interaction is mechanically robust Público Deposited

https://scholar.colorado.edu/concern/undergraduate_honors_theses/3t945r31n
Abstract
  • Protein-ligand interactions govern essential and ubiquitous biological processes such as immune response and gene regulation. Recently, the first computationally designed ligand-binding protein named DIG10.3 was developed by the Baker lab at the University of Washington. This artificially designed (rather than naturally evolved) ligand binding protein exhibited high affinity and selectivity to its target ligand, Digoxigenin (Dig). Such computationally designed ligand-binders offer promising capabilities in diagnostics and therapeutics for a wide range of diseases. By applying a mechanical force to a single DIG10.3::Digoxigenin interaction through atomic force microscope (AFM)-based single-molecule force spectroscopy (SMFS) we can extract unique information on the energy landscape which describes the interaction. This information consists of the distance to the transition state, the intrinsic off-rate, and the free energy of activation. To successfully study DIG10.3::Dig through AFM-based SMFS, improvements in biomolecular surface coupling techniques and in geometric correction of AFM measurements needed to be developed. We demonstrate that the DIG10.3::Dig interaction is comparable in stability to the analogous antibody-ligand interaction anti-dig::Dig. Therefore, DIG10.3 can serve as a cost-efficient alternative to anti-dig for SMFS studies since DIG10.3 can be expressed in E. Col. Finally, we expect such single-molecule studies of computationally designed ligand-binding proteins to facilitate the protein design process by providing iterative feedback on the mechanical strength of a protein-ligand interaction to protein engineers.
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Date Awarded
  • 2016-01-01
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Última modificación
  • 2019-12-02
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