An efficient high-throughput protocol based on 2D-HN(C)N for unambiguous H^N and ¹⁵N backbone assignment in small folded proteins in less than a day

Abstract

An efficient high-throughput method for sequential assignment of backbone ^lH^N and ^l5N, atoms in less than a day (using 2-4 2D spectra; total data collection in 4-8 h) has been proposed here. This is based on sequential correlations and specific patterns of peaks around the glycines, alanines, serines/threonines (internal check points) observable in the F_l-F₃ projection planes of the 3D-HN(C)N spectral variants. The F₂-F₃ projection planes of the spectra provide unique identification of the check Eoints. The protocol has been demonstrated on two ¹³C/^l5N_labelled proteins: ubiquitin and calbindin-D9k. In either case complete H^N and ^l5N backbone assignments were obtained in less than a day each. The method would be valuable for NMR structural studies of small, well-folded proteins.