  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.
