hNCOcanH pulse sequence and a robust protocol for rapid and unambiguous assignment of backbone (1HN, 15N and 13C′) resonances in 15 N/13C‐labeled proteins

Media type: E-Article
Title: hNCOcanH pulse sequence and a robust protocol for rapid and unambiguous assignment of backbone (1HN, 15N and 13C′) resonances in 15 N/13C‐labeled proteins
Contributor: Kumar, Dinesh; Hosur, Ramakrishna V.
imprint: Wiley, 2011
Published in: Magnetic Resonance in Chemistry
Language: English
DOI: 10.1002/mrc.2787
ISSN: 0749-1581; 1097-458X
Keywords: General Materials Science ; General Chemistry
Origination:
Footnote:
Description: <jats:title>Abstract</jats:title><jats:p>A three‐dimensional nuclear magnetic resonance (NMR) pulse sequence named as hNCOcanH has been described to aid rapid sequential assignment of backbone resonances in <jats:sup>15</jats:sup>N/<jats:sup>13</jats:sup>C‐labeled proteins. The experiment has been derived by a simple modification of the previously described HN(C)N pulse sequence [Panchal <jats:italic>et al.</jats:italic>, J. Biomol. NMR 20 (2001) 135–147]; <jats:italic>t</jats:italic><jats:sub>2</jats:sub> evolution is used to frequency label <jats:sup>13</jats:sup>C′ rather than <jats:sup>15</jats:sup>N (similar trick has also been used in the design of hNCAnH pulse sequence from hNcaNH [Frueh <jats:italic>et al.</jats:italic>, JACS, 131 (2009) 12880–12881]). The modification results in a spectrum equivalent to HNCO, but in addition to inter‐residue correlation peaks (i.e. H<jats:sub><jats:italic>i</jats:italic></jats:sub>, C<jats:sub><jats:italic>i</jats:italic>−1</jats:sub>), the spectrum also contains additional intra‐residue correlation peaks (i.e. H<jats:sub><jats:italic>i</jats:italic>−1</jats:sub>, C<jats:sub><jats:italic>i</jats:italic>−1</jats:sub>) in the direct proton dimension which has maximum resolution. This is the main strength of the experiment and thus, even a small difference in amide <jats:sup>1</jats:sup>H chemical shifts (5–6 Hz) can be used for establishing a sequential connectivity. This experiment in combination with the HNN experiment described previously [Panchal <jats:italic>et al.</jats:italic>, J. Biomol. NMR 20 (2001) 135–147] leads to a more robust assignment protocol for backbone resonances (<jats:sup>1</jats:sup>H<jats:sup>N</jats:sup>, <jats:sup>15</jats:sup>N) than could be derived from the combination of HNN and HN(C)N experiments [Bhavesh <jats:italic>et al.</jats:italic>, Biochemistry, 40 (2001) 14727–14735]. Further, this new protocol enables assignment of <jats:sup>13</jats:sup>C′ resonances as well. We believe that the experiment and the protocol presented here will be of immense value for structural—and functional—proteomics research by NMR. Performance of this experiment has been demonstrated using <jats:sup>13</jats:sup>C/<jats:sup>15</jats:sup>N labeled ubiquitin. Copyright © 2011 John Wiley & Sons, Ltd.</jats:p>

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