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Nuclear Magnetic Resonance Spectroscopy Studies of At2g44920, a Pentapeptide Repeat Protein from Arabidopsis thaliana and X-ray Crystallography, Isothermal Titration Calorimetry Studies of K-Ras, a Human Oncogenic GTP-ase Signaling Protein

Xu, Shenyuan
http://orcid.org/0000-0002-9903-1944
http://rave.ohiolink.edu/etdc/view?acc_num=miami1500896385881469

Abstract Details

2017, Doctor of Philosophy, Miami University, Chemistry and Biochemistry.
At2g44920 from Arabidopsis thaliana is a pentapeptide-repeat protein (PRP) composed of 25 repeats capped by N- and C-terminal a-helices. PRP structures are dominated by four-sided right-handed ß-helices typically consisting of mixtures of type II and type IV ß-turns. PRPs adopt repeated five-residue (Rfr) folds with an Rfr consensus sequence (STAV)(D/N)(L/F)(S/T/R)(X). Unlike other PRPs, At2g44920 consists exclusively of type II ß-turns. Given its unusual structure, we investigated the biophysical properties of At2g44920 as a representative of the ß-helix family to determine if it had exceptional global stability, backbone dynamics or amide hydrogen exchange rates. At2g44920 exhibited a striking range of amide hydrogen exchange rates spanning ten orders of magnitude, with lifetimes ranging from minutes to several months. A weak correlation was found between hydrogen exchange rates, hydrogen bonding energies and amino acid solvent accessible areas. Analysis of contributions from fast (~ps-ns) backbone dynamics to amide hydrogen exchange rates revealed that the average order parameter of amides undergoing fast exchange was significantly smaller compared to those undergoing slow exchange. Importantly, the activation energies for amide hydrogen exchange were found to be generally higher for the slowest exchanging amides in the central Rfr coil and decreased towards the terminal coils. K-Ras are small guanine-nucleotide binding proteins that function as molecular switches regulating multiple signal transducing pathways responsible for cell growth. Interactions between K-Ras, guanine-nucleotide exchange factor and GTPase-activating protein (GAP) control switching between a K-Ras “inactive” state and its “active” state. GAP significantly accelerates intrinsic GTP hydrolysis of wild type K-Ras, however, GAPs are incompetent at catalyzing GTP hydrolysis with K-Ras containing G12 mutations, which are found in ~30% of all human cancers. To give insight about the cell transformation originating from this single amino acid substitution, we report the crystal structure of K-Ras G12A mutation at 1.35 Å. Also, we used isothermal titration calorimetry to study the thermodynamics of GAP binding to several K-Ras G12 mutants. The results indicated that GAP had significantly increased affinity for G12X-K-Ras-GTP complexes compared to wt-K-Ras-GTP complexe. It was hypothesized that GAP inserts its side chain of arginine-789 into the active site of K-Ras to stabilize the transition state during hydrolysis reaction, which is supported by ITC results that GAP binds to K-Ras G12 mutants are entropy favored. Finally, we want to engineer the arginine finger region of GAP protein to restore its functions in promoting GTP hydrolysis of K-Ras mutants.
Michael Kennedy (Advisor)
David Tierney (Committee Chair)
Carole Dabney-Smith (Committee Member)
Rick Page (Committee Member)
Paul Urayama (Committee Member)
183 p.
Biochemistry; Biophysics
AT2g44920; NMR; hydrogen exchange; dynamic; K-Ras; G12A-K-Ras mutant; ITC; X-Ray

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Citations

  • Xu, S. (2017). Nuclear Magnetic Resonance Spectroscopy Studies of At2g44920, a Pentapeptide Repeat Protein from Arabidopsis thaliana and X-ray Crystallography, Isothermal Titration Calorimetry Studies of K-Ras, a Human Oncogenic GTP-ase Signaling Protein [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1500896385881469

    APA Style (7th edition)

  • Xu, Shenyuan. Nuclear Magnetic Resonance Spectroscopy Studies of At2g44920, a Pentapeptide Repeat Protein from Arabidopsis thaliana and X-ray Crystallography, Isothermal Titration Calorimetry Studies of K-Ras, a Human Oncogenic GTP-ase Signaling Protein . 2017. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1500896385881469.

    MLA Style (8th edition)

  • Xu, Shenyuan. "Nuclear Magnetic Resonance Spectroscopy Studies of At2g44920, a Pentapeptide Repeat Protein from Arabidopsis thaliana and X-ray Crystallography, Isothermal Titration Calorimetry Studies of K-Ras, a Human Oncogenic GTP-ase Signaling Protein ." Doctoral dissertation, Miami University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=miami1500896385881469

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Miami University and OhioLINK.