Reduced dimensionality (4,3)D-HN(C)NH for rapid assignment of 1H(N)-15N HSQC peaks in proteins: an analytical tool for protein folding, proteomics, and drug discovery programs.

While nuclear magnetic resonance (NMR) has had commendable success in atomic-level investigation of folded proteins, intrinsically unfolded and partially folded proteins have always posed a great challenge, because of poor chemical shift dispersions. We present here a reduced-dimensionality-based NMR triple resonance pulse sequence, (4,3)D-HN(C)NH, which not only helps to disperse the peaks further by combining (15)N and amide (1)H chemical shifts, but also directly establishes correlations between (1)H(i)(N), (15)N(i), (1)H(i+1)(N), and (15)N(i+1) spins along the F(1)-F(3) planes. The F(2)-F(3) projection planes of this experiment provide unique identification of the check points in amide resonances. An assignment strategy derived by combining information along the F(1)-F(3) planes and in the F(2)-F(3) projection planes of the experiment has been presented and shown to be very useful for both intrinsically disordered/unfolded proteins and folded protein alike. The experiment and the protocol would be valuable for protein folding, proteomics, and drug discovery programs.