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Conformational analysis of E. coli DnaT and the complex with PriA N-terminal domain

Easthon, Lindsey

Abstract Details

2010, Master of Science, University of Toledo, Chemistry.
The fidelity of DNA replication is essential to maintaining genomic integrity. Therfore, pathways to restart DNA replication upon fork arrest are essential to an organism’s survival. In E. coli there are seven protein involved in the formation of the replication restart primosome, PriA, PriB, PriC, DnaT, DnaB, DnaC and SSB. To date, with the exception of PriB, there is very little structural information available for these proteins or the complexes formed during primosome assembly. Electrospray ionization mass spectrometry (ESI-MS) can provide a great amount of structural information, beyond a simple mass measurement. Since ESI-MS is a soft ionization technique it allows for investigation of proteins under native folding conditions as well as non-covalent complexes. Combination of ESI-MS with ion mobility spectroscopy and collision induced dissociation experiments provide information about the shape and size of the protein and stability of non-covalent complexes. This work focused on conformational analysis of E. coli DnaT and the N-terminal domain of PriA using ESI-MS. The oligomerization states of the proteins were determined under native conditions. For DnaT, metal binding studies, CID, IMS, and limited proteolysis studies were used to analyze the conformation of the proteins in solution. An attempt was made to investigate the complex formed between the N-terminal domain of PriA and DnaT but no complex was observed. Finally, a buffer screen was developed to determine volatile buffers and salts for metal binding studies suitable for use in ESI-MS experiments.
Timothy Mueser (Advisor)
Wendell Griffith (Advisor)
Donald Ronning (Committee Member)
113 p.

Recommended Citations

Citations

  • Easthon, L. (2010). Conformational analysis of E. coli DnaT and the complex with PriA N-terminal domain [Master's thesis, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1271434912

    APA Style (7th edition)

  • Easthon, Lindsey. Conformational analysis of E. coli DnaT and the complex with PriA N-terminal domain. 2010. University of Toledo, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1271434912.

    MLA Style (8th edition)

  • Easthon, Lindsey. "Conformational analysis of E. coli DnaT and the complex with PriA N-terminal domain." Master's thesis, University of Toledo, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1271434912

    Chicago Manual of Style (17th edition)