The present work consists of three parts. First, I demonstrate the effectiveness of C12E5/hexanol as an orienting media for the measurement of residual dipolar couplings (RDCs) at low pH and for proteins with the positively charged surfaces, such as carbon storage regulator A at pH 4.5 and catalytic fragment of 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP-CF; theoretical pI 8.99). Second, the structure of the regeneration-induced CNPase homolog was determined based on 15N-1 H, 13C'-13C alpha, and 13Calpha- 1Halpha RDCs measured in Pf1 phage alignment media. Finally, 2D experiments based on the recently introduced SAD-REDOR technique for the measurement of RDCs in proteins were developed. The SAD-REDOR (single-alignment domain rotational-echo double resonance) technique consists of magic angle spinning (MAS) and rotor synchronized radiofrequency pulses applied to the molecule fixed in the polymer-stabilized liquid crystalline media. MAS averages dipolar couplings to zero, while the application of the RF pulses allows selective recovery of chemical shift anisotropy, homonuclear or heteronuclear RDCs. SAD-REDOR can be used to study strongly aligned biomolecules. It allows measurement of both scalar and dipolar couplings in a single sample and control over their magnitudes. Here, I present SAD-IPAP and SAD-HSQC experiments for the measurements of 15N-1 H RDCs in direct and indirect dimensions, respectively, as it is demonstrated for 15N-enriched PSLC ubiquitin.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.98771 |
| Date | January 2005 |
| Creators | Pomerantseva, Ekaterina. |
| 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 Biochemistry.) |
| Rights | © Ekaterina Pomerantseva, 2005 |
| Relation | alephsysno: 002326500, proquestno: AAIMR24772, Theses scanned by UMI/ProQuest. |
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