Variable-temperature Fourier transform infrared (VT-FTIR) spectroscopy in conjunction with 2D correlation analysis was employed to study the unfolding of aspartate transcarbamylase (ATCase) and its individual subunits. The regulatory subunit (RSU) was uniformly labeled with 13C/15N and then reconstituted with the unlabeled catalytic subunit (CSU) to form the holoenzyme. The activity of the holoenzyme was shown to be unaffected by the isotopic labeling of the RSU. The VT-FTIR investigation of the isolated CSU and the CSU in the holoenzyme revealed that the CSU is more thermally stable when bound to the RSU (i.e., in the holoenzyme). The RSU also showed more thermal stability when bound to the CSU. The sequences of events leading to the unfolding of the isolated CSU and RSU and the CSU in the holoenzyme were deduced by 2D correlation analysis of the VT-FTIR spectra. The results for the isolated CSU demonstrated that beta-sheets unfold first, followed by a-helices and then turns, and finally aggregates form. The sequence of unfolding of the RSU showed an increase of turns followed by a loss of intramolecular beta sheets, then a loss of alpha-helices and the formation of aggregates. The CSU in the holoenzyme exhibited a slightly different unfolding pathway and was observed to unfold subsequent to the unfolding of the RSU, consistent with the two thermal transitions observed by differential scanning calorimetry.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.32999 |
| Date | January 2001 |
| Creators | Haque, Takrima. |
| Contributors | Ismail, Ashraf A. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Food Science and Agricultural Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001846615, proquestno: MQ75314, Theses scanned by UMI/ProQuest. |
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