Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.661314
Title: Use of an extended set of 3D NOESY experiments for the structure determination of double-labelled proteins by NMR
Author: Robinson, Philip Tobias
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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Abstract:
A method for increasing the efficiency of NMR is proposed whereby a set of 3D NOESY experiments which employ heteronuclear labelling in both indirect dimensions are used to aid assignment of standard 3D 15N- and 13C-edited NOESY spectra. Using these experiments in concert allows more precise identification of the 1H nuclei involved in NOE interactions and, in doing so, ambiguity in the assignment of NOESY peaks is significantly reduced or eliminated. The complementary experiments investigated are a 3D 13C, 15N-HSQC-NOESY-HSQC and 3D 15N, 15N-HSQC-NOESY-HSQC for use with 3D 15N-edited NOESY and 3D 13C, 13C-HSQC-NOESY-HSQC for use with 3D 13C-edited NOESY. A methyl selective experiment, 3D 13C, 13CH3-HSQC-NOESY-HSQC for use with 3D 13CH3-edited NOESY is also included. Pulse programs for these sequences have been written and implemented. Semi-automated assignment protocols, developed within the CCPNMR Analysis software package, are developed and used to allow effective manipulation and elucidation of the multiple spectra, streamlining the process. The procedure is tested on the 18 kDa protein, β-Lactoglobulin B and the 15 kDa protein C4BP~1,2. The extent to which assignment ambiguity is reduced relative to a standard assignment method and the effect upon the time requirements for structure determination is investigated. It is shown that, for the sizes of proteins investigated, the time needed to obtain solution structures is reduced from months to weeks. During the course of this work, a near-complete resonance assignment of the protein β-Lactoglobulin B is achieved using standard triple resonance experiments for backbone and sidechain assignment and the low pH solution structure of this protein is solved.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.661314  DOI: Not available
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