Pseudo 5D HN(C)N experiment to facilitate the assignment of backbone resonances in proteins exhibiting high backbone shift degeneracy

Assignment of protein backbone resonances is most routinely carried out using triple resonance three-dimensional NMR experiments involving amide ¹H/¹⁵N resonances. However for intrinsically unstructured proteins, alpha-helical proteins or proteins containing several disordered fragments, the assignment becomes problematic because of high-degree of backbone shift degeneracy. In this backdrop, a novel reduced-dimensionality (RD) experiment -(5, 3)D-hNCO-CANH- is presented to facilitate/validate the sequential backbone resonance assignment in such proteins. The proposed 3D NMR experiment makes use of the modulated amide ¹⁵N chemical shifts (resulting from the joint sampling along both its indirect dimensions) to resolve the ambiguity involved in connecting the neighboring amide resonances (i.e. H_iN_i and H_i-1N _i-1) for overlapping amide-NH peaks. The experiment -in combination with routine triple resonance 3D-NMR experiments involving backbone amide (¹H/¹⁵N) and carbon (¹³C ^α/¹³C′) chemical shifts- will serve as a powerful complementary tool to achieve the nearly complete assignment of protein backbone resonances in a time efficient manner.