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

Abstract

Abstract A three‐dimensional nuclear magnetic resonance (NMR) pulse sequence named as hNCOcanH has been described to aid rapid sequential assignment of backbone resonances in 15 N/ 13 C‐labeled proteins. The experiment has been derived by a simple modification of the previously described HN(C)N pulse sequence [Panchal et al. , J. Biomol. NMR 20 (2001) 135–147]; t 2 evolution is used to frequency label 13 C′ rather than 15 N (similar trick has also been used in the design of hNCAnH pulse sequence from hNcaNH [Frueh et al. , 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 i , C i −1 ), the spectrum also contains additional intra‐residue correlation peaks (i.e. H i −1 , C i −1 ) 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 1 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 et al. , J. Biomol. NMR 20 (2001) 135–147] leads to a more robust assignment protocol for backbone resonances ( 1 H N , 15 N) than could be derived from the combination of HNN and HN(C)N experiments [Bhavesh et al. , Biochemistry, 40 (2001) 14727–14735]. Further, this new protocol enables assignment of 13 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 13 C/ 15 N labeled ubiquitin. Copyright © 2011 John Wiley & Sons, Ltd.