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Measuring C-13(beta) chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
Hospital for Sick Children, Toronto.
University of Toronto.
2009 (English)In: JOURNAL OF BIOMOLECULAR NMR, ISSN 0925-2738, Vol. 44, no 3, 139-155 p.Article in journal (Refereed) Published
Abstract [en]

A labeling scheme is introduced that facilitates the measurement of accurate C-13(beta) chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of C-13 enrichment (30-40%) at C-beta side-chain carbon positions for 15 of the amino acids with little C-13 label at positions one bond removed (a parts per thousand 5%). A pair of samples are produced using [1-C-13]-glucose/(NaHCO3)-C-12 or [2-C-13]-glucose as carbon sources with isolated and enriched (andgt; 30%) C-13(beta) positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of C-13(beta) chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein-ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples.

Place, publisher, year, edition, pages
2009. Vol. 44, no 3, 139-155 p.
Keyword [en]
CPMG, C-13(beta) Chemical shifts, Selective labeling, Chemical exchange, Excited protein states
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-19621DOI: 10.1007/s10858-009-9321-3OAI: diva2:226726
Available from: 2009-07-03 Created: 2009-07-03 Last updated: 2009-07-03

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Lundström, Patrik
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Molecular Biotechnology The Institute of Technology
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ReferencesLink to record
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