Rapid and efficient methods for preparing isotopically labeled recombinant proteins and refining solved structures via NMR are presented. The former approach was developed for 2H/13C/15N isotopic labeling and specific protonation of the methyl groups of isoleucine, leucine, and valine (ILV) residues of rat brain calbindin D28K, a calcium sensor and buffer. This protocol produces cell mass using unlabeled rich media followed by exchange into labeled media at high cell density. Allowing for a short period for growth recovery and unlabeled metabolite clearance, the cells were induced. Additionally, the solution structure of LuxU, a subunit of the quorum sensing circuit of Vibrio harveyi, has been refined using residual dipolar coupling (RDC). In slightly anisotropic environments, large one-bond internuclear dipolar interactions no longer average to zero, and therefore can provide information on the average orientation of the corresponding vectors relative to the magnetic field. This ordering was induced by introducing Pf1 filamentous bacteriophage into a solution of 13C15N isotopically labeled LuxU.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-03282004-195953 |
| Date | 01 April 2004 |
| Creators | Rogers, Constance Ann |
| Contributors | John Cavanagh, William Miller, Michael Goshe |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-03282004-195953/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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