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 â 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, âExperimental and Computational Identification of Naïve Binders to a TIM-Barrel Protein Scaffoldâ (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, âComputational Design of Protein-Small Molecule Interfacesâ (first author). A detailed description of how to utilize RosettaLigand is the focus of Chapter 4, âRosetta and Design of Ligand Binding Sitesâ (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 âDesigned C9S_HisF Binds VU0068924 More Tightlyâ, 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.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03282016-022145 |
| Date | 07 April 2016 |
| Creators | Allison, Brittany Ann |
| Contributors | Jens Meiler, Ph.D., Brian O. Bachmann, Ph.D., Michael P. Stone, PhD., John A. Capra, Ph.D. |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-03282016-022145/ |
| Rights | restrictone, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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