Direct Sequential Hit Strategy for Unambiguous and Accurate Backbone Assignment of 13C/15N Labeled Proteins

Abstract

Backbone assignment is the very first and key requirement of any study of protein by NMR. However, the process is generally made difficult by severe amide shift degeneracy commonly exhibited by higher MW (>15 kDa in size) alpha-helical and structurally disordered proteins. Pulse sequences and novel strategies have been reported earlier relying heavily on well dispersed nitrogen chemical shifts. In this context, we present here extension of HNN and HN(C)N based assignment protocol (Panchal et al., J Biomol NMR 20:135–147, 2001) by exploiting carbon chemical shifts to resolve the ambiguities arising because of amide 15N/1H shift degeneracy. The resulted modifications have been termed as hNCAnH and hNcoCAnH, respectively and based on the complementary and mutually exclusive information contained in these two spectra, an efficient strategy -named here as strategy—has been presented for unambiguous and accurate backbone assignment of 13C/15N labeled proteins. The strategy provides directly the coordinates of sequential amide peaks (H i−1, N i−1 and H i+1, N i+1) for a particular amide cross peak (H i , N i ), just after performing the simple manual peak picking step. Thus, the sequential (i to i − 1 or i to i + 1) connectivities between the backbone amide cross peaks (H i , N i ) are established directly without making cumbersome search through various planes of the 3D spectrum as is the case with other presently used approaches for backbone assignment. The strategy have been demonstrated here using an intrinsically unfolded 164-residue protein named DLC1 binding domain of nNOS (residues 134–298).