Abstract
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 14NN(CO)[CAHA] experiments, the 13Cα and 1Hα 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/1HN/13Cα or 15N/1HN/1Hα, 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 HNN CACO, where 13Cα and 13C′ chemical shifts evolve in a common dimension. The 13Cα 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.
Original language | English (US) |
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Pages (from-to) | 41-50 |
Number of pages | 10 |
Journal | Journal of biomolecular NMR |
Volume | 24 |
Issue number | 1 |
DOIs | |
State | Published - Sep 1 2002 |
Bibliographical note
Funding Information:This work was supported by the Ontario Research and Development Challenge Fund (to C.H.A.), the National Science Foundation (MCB 0075773 to T.S.) and the National Institutes of Health (Northeast Structural Genomics Consortium; P50 GM62413-01 to T.S. and C.H.A.). We thank Dr A. Yee and A. Semesi for helpful discussions.
Keywords
- Automated protein NMR assignment
- Protein structure
- Reduced-dimensionality triple-resonance experiments
- Resolution enhancement
- Structural genomics