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

A three-dimensional nuclear magnetic resonance (NMR) pulse sequence named as hNCOcanH has been described to aid rapid sequential assignment of backbone resonances in ¹⁵N/¹³C-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]; t₂ evolution is used to frequency label ¹³C' rather than ¹⁵N (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 ¹H 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 (¹H^N, ¹⁵N) 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 ¹³C' 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 ¹³C/¹⁵N labeled ubiquitin.