Fractional enrichment of proteins using [2-C-13]-glycerol as the carbon source facilitates measurement of excited state C-13 alpha chemical shifts with improved sensitivity
2015 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 62, no 3, 341-351 p.Article in journal (Refereed) Published
A selective isotope labeling scheme based on the utilization of [2-C-13]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state C-13 alpha chemical shifts using Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the C alpha positions is increased two-fold relative to labeling schemes based on [2-C-13]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state C-13 alpha chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s(-1), despite the small fraction of C-13 alpha-C-13 beta spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using C-13 alpha spin probes.
Place, publisher, year, edition, pages
Springer Verlag (Germany) , 2015. Vol. 62, no 3, 341-351 p.
CPMG; C-13 alpha labeling; [2-C-13]-Glycerol; Excited states
IdentifiersURN: urn:nbn:se:liu:diva-120348DOI: 10.1007/s10858-015-9948-1ISI: 000357489200010PubMedID: 25990019OAI: oai:DiVA.org:liu-120348DiVA: diva2:843890
Funding Agencies|Swedish Research Council [2012-5136]2015-07-312015-07-312015-08-06