Reduced Dimensionality tailored HN(C)N Pulse Sequences for Efficient Backbone Resonance Assignment of Proteins through Rapid Identification of Sequential HSQC peaks

Abstract

Two novel reduced dimensionality (RD) experiments -(4,3)D-hNCOcaNH and (4,3)D-hNcoCANH- have been presented here to facilitate the backbone resonance assignment of proteins both in terms of its accuracy and speed. The experiments basically represent an improvisation of previously reported HN(C)N experiment [Panchal et. al., J. Biomol. NMR. (2002), 20 (2), 135-147] and exploit the simple reduced dimensionality NMR concept [Szyperski et. al. (2002), Proc. Natl. Acad. Sci. U.S.A. 99(12), 8009-8014] to achieve (a) higher dispersion and resolution along the co-evolved F1 dimension and (b) rapid identification of sequential HSQC peaks on its F2(15N)- F3(1H) planes. The current implementation is based on the fact that the linear combination of 15N and 13CO/13Ca chemical shifts offers relatively better dispersion and randomness compared to the individual chemical shifts; thus enables the assignment of crowded HSQC spectra by resolving the ambiguities generally encountered in HNCN based assignment protocol because of amide 15N shift degeneracy. Additionally, each of these experiments enables assignment of backbone 13CO/13Ca resonances as well. Overall, the reduced dimensionality tailored HNCN experiments presented here will be of immense value for various structural and functional proteomics studies by NMR; particularly of intrinsically or partially/unstructured proteins and medium sized (MW ~12-15 kDa) folded proteins. The experiments, like any other experiment that yields protein assignments, would be extremely valuable for protein folding and drug discovery programs as well.