Novel projected 4D triple resonance experiments for polypeptide backbone chemical shift assignment

Here we present a novel suite of projected 4D triple-resonance NMR experiments for efficient sequential assignment of polypeptide backbone chemical shifts in C-13/N-15 doubly labeled proteins. In the 3D HNN[CAHA] and 3D HNN(CO)[CAHA] experiments, the C-13(alpha) and H-1(alpha) chemical shifts evolve in a common dimension and are simultaneously detected in quadrature. These experiments are particularly useful for the assignment of glycine-rich polypeptide segments. Appropriate setting of the H-1 radiofrequency carrier allows one to place cross peaks correlating either backbone N-15/H-1(N)/C-13(alpha) or N-15/H-1(N) /H-1(alpha) chemical shifts in separate spectral regions. Hence, peak overlap is not increased when compared with the conventional 3D HNNCA and HNN( CA) HA. 3D HNN[ CAHA] and 3DHNN(CO)[CAHA] are complemented by 3D reduced-dimensionality (RD) HNN COCA and HNN CACO, where C-13(alpha) and C-13' chemical shifts evolve in a common dimension. The C-13(alpha) shift is detected in quadrature, which yields peak pairs encoding the C-13' chemical shift in an in-phase splitting. This suite of four experiments promises to be of value for automated high-throughput NMR structure determination in structural genomics, where the requirement to independently sample many indirect dimensions in a large number of NMR experiments may prevent one from accurately adjusting NMR measurement times to spectrometer sensitivity.