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Thesis_Callahan_Biophysics_Fall2014.pdf (5.29 MB)

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Bioinformatics-Driven Enzyme Engineering: Work On Adenylate Kinase

Callahan, Nicholas
http://rave.ohiolink.edu/etdc/view?acc_num=osu1420802270

Abstract Details

2015, Doctor of Philosophy, Ohio State University, Biophysics.
Despite the large databases of protein sequence and structure data presently available, predicting the effect of mutations on an enzyme is still a major challenge. Isolating and interpreting evidence in sequence alignments of major evolutionary events is one way to probe the chemical logic of enzymes' functional plasticity. In this thesis, I outline work on predicting mutations of structural significance in proteins using bioinformatic techniques and the characterization of said mutations in functional enzymes. For this work, I use the well-studied enzyme adenylate kinase (ADK) as my model system. In Chapter 1, I review the history and current research in bioinformatics-driven mutation prediction and chemical, biological, and biophysical aspects of adenylate kinase. Chapter 2 presents a series of bacterial adenylate kinase constructs which demonstrate key transitional variants, predicted from an alignment of ADKs, bridging functional ADK from B. subtilis and E. coli. Significantly, characterization of these variants has found necessary pre-conditions for the evolutionary elimination of a zinc binding site from B. subtilis ADK that is otherwise required for function. Chapter 3 presents the algorithmic implementation of an improved protocol for the prediction of consensus mutations, previously discussed by Dr. Brandon Sullivan and Dr. Thomas Magliery, and several consensus constructs of adenylate kinase, triosephosphate isomerase, and the DNA binding domain of fructose repressor FruR. Many of these constructs show thermal stabilization and native-like function, revealing the robustness of the new protocol. Additionally, I analyze of the common features across both our successful and unsuccessful constructs. Chapter 4 outlines preliminary work towards the further biophysical characterization of the ADK transitional variants discussed in chapter 2. Chapter 5 presents our attempts to produce constructs from key features of the human dp71 gene product, for the purpose of characterizing mutations implicated in Duchenne muscular dystrophy.
Thomas Magliery, Dr. (Advisor)
William Ray, Dr. (Committee Member)
Ralf Bundschuh, Dr. (Committee Member)
Amanda Bird, Dr. (Committee Member)
165 p.
Biophysics
Protein engineering; correlation; co-mutation; consensus proteins; Adenylate kinase

Recommended Citations

Citations

  • Callahan, N. (2015). Bioinformatics-Driven Enzyme Engineering: Work On Adenylate Kinase [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1420802270

    APA Style (7th edition)

  • Callahan, Nicholas. Bioinformatics-Driven Enzyme Engineering: Work On Adenylate Kinase. 2015. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1420802270.

    MLA Style (8th edition)

  • Callahan, Nicholas. "Bioinformatics-Driven Enzyme Engineering: Work On Adenylate Kinase." Doctoral dissertation, Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1420802270

    Chicago Manual of Style (17th edition)

osu1420802270
142
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This open access ETD is published by The Ohio State University and OhioLINK.