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Dissertation for Natalie Mikita.pdf (3.69 MB)

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DEVELOP SPECTROSCOPIC APPROACHES TO STUDY NON-PROTEOSOMAL ATP-DEPENDENT PROTEOLYSIS

Mikita, Natalie
http://rave.ohiolink.edu/etdc/view?acc_num=case1401814273

Abstract Details

2014, Doctor of Philosophy, Case Western Reserve University, Chemistry.
Lon is an ATP-dependent serine protease found ubiquitously in nature that degrades damaged, misfolded, and certain regulatory proteins in the cell while hydrolyzing ATP to ADP and inorganic phosphate. Using a physiological substrate of Esherichia coli Lon (ELon) protease known as the lambda N protein,and truncated mutants, it was demonstrated that the N-terminus is not important for efficient degradation by ELon, however the C-terminus is important in binding and degradation. Using fluorescent substrates of Lon, steady-state and pre-steady-state kinetic techniques were employed to determine the timing of scissile sites approaching the protoelytic site of ELon and their subsequent cleavages in an ATP- and AMPPNP- dependent manner. Collectively, these results support a mechanism by which ELon delivers lambda N to the active site C-terminally first, followed by the N-terminal. Subsequent cleavage awaits the almost complete delivery of all the sites, which appears to lack a specific directionality. Using hydrogen-deuterium exchange mass spectrometry (HXMS), deuterium incorporation in ELon was compared to known crystal structures solved. These results identified regions that corroborated the crystal structure, or differed from it. Previously developed assays were used to characterize twelve mutants of human Lon (hLon) implicated in causing cerebral, ocular, dental, auricular, and skeletal syndrome. The results of these assays so far does not indicate a clear mode of disease.
Irene Lee (Advisor)
Mary Barkley (Committee Chair)
Blanton Tolbert (Committee Member)
Yanming Wang (Committee Member)
Patrick Wintrode (Committee Member)
144 p.
Biochemistry; Chemistry
Protease; proteolysis; hydrogen-deuterium exchange; substrate translocation, ATP hydrolysis;

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Citations

  • Mikita, N. (2014). DEVELOP SPECTROSCOPIC APPROACHES TO STUDY NON-PROTEOSOMAL ATP-DEPENDENT PROTEOLYSIS [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1401814273

    APA Style (7th edition)

  • Mikita, Natalie. DEVELOP SPECTROSCOPIC APPROACHES TO STUDY NON-PROTEOSOMAL ATP-DEPENDENT PROTEOLYSIS. 2014. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1401814273.

    MLA Style (8th edition)

  • Mikita, Natalie. "DEVELOP SPECTROSCOPIC APPROACHES TO STUDY NON-PROTEOSOMAL ATP-DEPENDENT PROTEOLYSIS." Doctoral dissertation, Case Western Reserve University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1401814273

    Chicago Manual of Style (17th edition)

case1401814273
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© 2014, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.