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PROBING PROTEIN-PROTEIN INTERACTIONS in vitro and in vivo WITH CYANOGEN

WINTERS, MICHAEL SHAWN

Abstract Details

2002, PhD, University of Cincinnati, Arts and Sciences : Chemistry.
Salt bridges between self-associating hen egg-white (HEW) lysozyme and bovine insulin molecules were converted to covalent links by ethanedinitrile (cyanogen) and identified using mass spectrometry. Peptides resulting from cyanogen-mediated intermolecular cross-linking of HEW lysozyme were detected using in-gel digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sequence data from electrospray ionization quadrupole-time-of-flight mass spectrometry (ESI-Q-TOF MS) revealed that one of the peptides has a covalent bond between Asp 66 and Arg 14. Human lysozyme is one of twenty known proteins that form amyloid fibrils, a characteristic feature of systemic amyloidosis. The reported cases of lysozyme amyloidosis result from single point mutations in the lysozyme gene. One of the variant proteins contains a mutation at Asp67His (corresponding to Asp66His in HEW lysozyme), the same amino acid residue identified cross-linked in native HEW lysozyme associations using cyanogen. The self-assembly of another amyloid fibril protein, bovine insulin, was also investigated using cyanogen. Using high performance liquid chromatography (HPLC) coupled with ESI-Q-TOF MS, an intermolecular salt bridge association was identified by covalently linking the B chain C-terminal carboxyl group of Ala 30 and the charged imidazole of His 5 (B chain). These two residues are not known to associate closely in native insulin self-assembly. However a recent model of insulin fibril formation, proposed by Brange and colleagues, places these two residues in close intermolecular proximity. A method was developed incorporating enzymatic digestion, 1D gel electrophoresis, MALDI-TOF MS, and HPLC ESI-Q-TOF MS to identify amino acid residues participating in salt bridge formations at protein-protein interfaces. The same methodology was applied to protein-protein associations in Bacillus subtilis . Computer based searching and HPLC ESI-Q-TOF MS sequence data confirmed the identity of a group of ribosomal proteins cross-linked by cyanogen. The following in vivo cross-linked species were identified: L18-L4, L14-L31-L19, S4-S13, S12-L16, S11-initiation factor 3 (IF3), and pseudouridine synthase-L35 (L33). Nonribosomal proteins pseudouridine synthase and IF3 interact with the ribosome only at certain times in Bacillus subtilis cell cycle. The results presented in thesis show that this methodology can be applied to protein-protein interactions in the intact cell and also suggests the possibility of using cyanogen to tract cell cycle dependent events.
Dr. Richard A. Day (Advisor)
119 p.

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Citations

  • WINTERS, M. S. (2002). PROBING PROTEIN-PROTEIN INTERACTIONS in vitro and in vivo WITH CYANOGEN [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1027090541

    APA Style (7th edition)

  • WINTERS, MICHAEL. PROBING PROTEIN-PROTEIN INTERACTIONS in vitro and in vivo WITH CYANOGEN. 2002. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1027090541.

    MLA Style (8th edition)

  • WINTERS, MICHAEL. "PROBING PROTEIN-PROTEIN INTERACTIONS in vitro and in vivo WITH CYANOGEN." Doctoral dissertation, University of Cincinnati, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1027090541

    Chicago Manual of Style (17th edition)