  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.
