Reduced Dimensionality (4,3)D-hnCOCANH Experiment: An Efficient Backbone Assignment tool for NMR studies of Proteins

TL;DR

This paper introduces a new efficient NMR method, (4,3)D-hnCOCANH, for rapid backbone resonance assignment and secondary structure determination in proteins, simplifying data analysis and improving spectral dispersion.

Contribution

The method employs a single reduced dimensionality experiment that distinguishes intra- and inter-residue peaks and uses unidirectional correlations for robust, rapid backbone assignment.

Findings

Achieves better spectral dispersion using chemical shift combinations.

Allows direct distinction of intra- and inter-residue peaks without extra experiments.

Enables rapid, robust backbone assignment for structural proteomics.

Abstract

Sequence specific resonance assignment and secondary structure determination of proteins form the basis for variety of structural and functional proteomics studies by NMR. In this context, an efficient standalone method for rapid assignment of backbone (1H, 15N, 13Ca and 13C') resonances and secondary structure determination of proteins has been presented here. Compared to currently available strategies used for the purpose, the method employs only a single reduced dimensionality (RD) experiment -(4,3)D-hnCOCANH and exploits the linear combinations of backbone (13Ca and 13C') chemical shifts to achieve a dispersion relatively better compared to those of individual chemical shifts (see the text) for efficient and rapid data analysis. Further, the experiment leads to the spectrum with direct distinction of self (intra-residue) and sequential (inter-residue) carbon correlation peaks; these appear opposite in signs and therefore can easily be discriminated without using an additional complementary experiment. On top of all this, the main strength of the method is its efficient and robust assignment strategy based on: (a) multiple, but unidirectional sequential 15N and 13C correlations (i-->i-1) and (b) the distinctive peak patterns of self and sequential peaks in different [(F1(13C)-F3(1H) and (F2(15N)-F3(1H)] planes of the 3D spectrum which enable ready identification of certain specific triplet sequences (see the text) and thus serve as check points for mapping the stretches of sequentially connected HSQC cross peaks on to the primary sequence for assigning the resonances sequence specifically. Overall, the standalone method presented here will be an important backbone assignment tool for structural and functional proteomics, protein folding, and drug discovery research programs by NMR.