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Combinatorial library approach to decipher the stability effects of loop mutagenesis in model protein scaffold via high throughput methods

Please use this identifier to cite or link to this item: http://hdl.handle.net/1811/51768

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Title: Combinatorial library approach to decipher the stability effects of loop mutagenesis in model protein scaffold via high throughput methods
Creators: Sen, Shiladitya
Advisor: Magliery, Thomas J.
Issue Date: 2012-02
Abstract: The inability to accurately predict the relationship between protein’s primary sequence and its overall stability presents a major hurdle in determining the effects of mutations on protein folding. To study the effects of mutation, combinatorial libraries that involve randomizing many selected positions of a protein simultaneously, offer a broader, statistically relevant dataset. In this study, we have analyzed this complicated sequence-stability relationship using rigorous high-throughput (HT) methods. Our studies focus on the four-helix model bundle protein Rop through the means of combinatorial repacking the loop. The loop was randomized with and without an additional residue to make it more flexible. Using a novel screen we identified functional variants of Rop that were of similar or higher in stability than the parent scaffold. HT biophysical characterization and detailed Gibbs-Helmholtz studies were carried out to probe the entropy and enthalphy contribution on stability for selected variants. A potential salt bridge interaction was necessary to enhance the stability of the protein without affecting its function or fold.
Embargo: A five-year embargo was granted for this item.
Series/Report no.: 2012. Edward F. Hayes Graduate Research Forum. 26th
Keywords: Combinatorial
High-throughput
Stability
Loop mutagenesis
Gibbs-Helmholtz
protein folding
Description: Mathematical and Physical Sciences: 3rd Place (The Ohio State University Edward F. Hayes Graduate Research Forum)
URI: http://hdl.handle.net/1811/51768
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