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 and 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 (and/or to 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. HiNi and Hi-1Ni-1) for overlapping amide NH peaks. The experiment -encoding 5D spectral information- leads to a conventional 3D spectrum with significantly reduced spectral crowding and complexity. The improvisation is based on the fact that the linear combinations of intra-residue and inter-residue backbone chemical shifts along both the co-evolved indirect dimensions span a wider spectral range and produce better peak dispersion than the individual shifts themselves. Taken together, the experiment -in combination with routine triple resonance 3D NMR experiments involving backbone amide (1H and 15N) and carbon (13C-alpha and 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. The performance of the experiment and application of the method have been demonstrated here using a 15.4 kDa size folded protein and a 12 kDa size unfolded protein. 
Dinesh Kumar, Nisha Raikwal, Vaibhav Kumar Shukla, Himanshu Pandey,   Ashish Arora and Anupam Guleria
physics.chem-ph physics.bio-ph q-bio.BM