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 13C/15N doubly labeled proteins. In the 3D HNN[CAHA] and 3D HNN(CO)[CAHA] experiments. the 13C(alpha) and lHalpha 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 1H radiofrequency carrier allows one to place cross peaks correlating either backbone 15N/1H(N)/13C(alpha) or 15N/1H(N)/H(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 3D HNN(CO)[CAHA] are complemented by 3D reduced-dimensionality (RD) HNN COCA and NNCACO, where 13C(alpha) and 13C' chemical shifts evolve in a common dimension. The 13C(alpha), shift is detected in quadrature, which yields peak pairs encoding the 13C' 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.
Xia
Youlin
Y
Ontario Cancer Institute and Department of Medical Biophysics, The University of Toronto, Canada.
Arrowsmith
Cheryl H
CH
Szyperski
Thomas
T
D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation
D010455:Peptides