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Understanding Protein Structure And Function Using Rational Design And Unnatural Amino Acids

Mata, David Garcia
http://rave.ohiolink.edu/etdc/view?acc_num=osu1338392020

Abstract Details

2012, Doctor of Philosophy, Ohio State University, Chemistry.
Rational design is a powerful tool that can be used to understand the function of a protein, enhance a desired function, or engineer novel functionalities entirely. To this end, we have employed rational design toward the engineering of paraoxonase-1 (PON1). PON1 is able to hydrolyze a broad range of substrates, including toxic organophosphorus (OP) compounds at low levels, which makes it an attractive candidate for rational design. As the means by which PON1 is able to hydrolyze such a broad range of substrates is unknown, we investigated mechanistically-relavant residues along the active site and calcium coordination sphere. While our results mostly support previously proposed mechanistic hypotheses, it appears that hydrolysis of OP substrates may be more complex than originally believed. To provide a foundation for future iterations of rational design, we constructed a comprehensive catalog of mutations along the PON1 active site with the goal of rationalizing substrate specificity. Using the knowledge attained from this study as an input, we constructed rationally-designed directed libraries of PON1. Selected library variants displayed increased activity towards the toxic isomer of GF, further demonstrating the power of rational design. Finally, we report incorporation of unnatural amino acids p-Benzoylphenylalanine, o-nitrobenzyltyrosine, hydroxyquinolinylalanine and bipyridylalanine into proteins in order to rationally incorporate novel functionalities into proteins.
Thomas Magliery, PhD (Advisor)
Christopher Hadad, PhD (Committee Member)
Christopher Jaroniec, PhD (Committee Member)
Vijay Pancholi, PhD (Committee Member)

Recommended Citations

Citations

  • Mata, D. G. (2012). Understanding Protein Structure And Function Using Rational Design And Unnatural Amino Acids [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338392020

    APA Style (7th edition)

  • Mata, David. Understanding Protein Structure And Function Using Rational Design And Unnatural Amino Acids. 2012. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1338392020.

    MLA Style (8th edition)

  • Mata, David. "Understanding Protein Structure And Function Using Rational Design And Unnatural Amino Acids." Doctoral dissertation, Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338392020

    Chicago Manual of Style (17th edition)

osu1338392020
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© 2012, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.