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Title page for ETD etd-03282016-022145

Type of Document Dissertation
Author Allison, Brittany Ann
URN etd-03282016-022145
Title Computational Design of Protein-Ligand Interfaces Using RosettaLigand
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Jens Meiler, Ph.D. Committee Chair
Brian O. Bachmann, Ph.D. Committee Member
John A. Capra, Ph.D. Committee Member
Michael P. Stone, PhD. Committee Member
  • protein engineering
  • protein ligand binding
  • RosettaLigand
  • Rosetta
  • protein small molecule interactions
  • interface design
  • computational design
  • ligand macromolecule recognition
  • NMR
  • binding affinity
Date of Defense 2016-03-11
Availability unrestricted
Computational design of protein-ligand interfaces expands understanding of the basic forces involved in molecular recognition, and also contributes to the development of protein therapeutics. My dissertation research contributes to this body of knowledge through a series of Specific Aims. Specific Aim 1 involves screening a diverse set of small molecules for intrinsic binding affinity to my protein, HisF. 28 binding ligands were identified by using nuclear magnetic resonance (NMR) techniques by tracking chemical shift peaks. This also allows us to calculation dissociation constants, which ranged between 340 – 1110 µM. These binding ligands were then computationally docked into HisF using RosettaLigand of the Rosetta modeling suite. Computational results were compared to the experimental data to identify strengths/weaknesses of the program. These results are the focus of Chapter 3, “Experimental and Computational Identification of Naïve Binders to a TIM-Barrel Protein Scaffold” (first author), to be submitted soon. Specific Aim 2 involved optimizing RosettaLigand to design proteins that bind small molecules. The software was tested for accuracy and efficiency using a set of protein-ligand crystal structures, and these results are the focus of my 2014 published manuscript and Chapter 2, “Computational Design of Protein-Small Molecule Interfaces” (first author). A detailed description of how to utilize RosettaLigand is the focus of Chapter 4, “Rosetta and Design of Ligand Binding Sites” (secondary author), manuscript accepted. Specific Aim 3 combines the first two aims, to redesign the protein interface to bind the small molecules more tightly than the wild type protein. We have used RosettaLigand to redesign HisF to bind one VU0068924 more tightly, with binding affinity improving from 442 µM to 23 µM. This is the focus of Appendix C “Designed C9S_HisF Binds VU0068924 More Tightly”, and will be the focus of a future manuscript. For each project, the protocols, scripts, command-lines, experiments not described in the manuscript are included in the appendix. The models, code, scripts, and figures are included in the thesis directory that accompanies the thesis.
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