Automated Resonance Assignment of Proteins: 6 DAPSY-NMR

The 6-dimensional(6D) APSY-seq-HNCOCANH NMR experiment correlates two sequentially neighbor in gamidemoieties in proteins via the C′ and C(α) nuclei, with efficient suppression of the back transfer from C(α) to the originating amidemoiety. The automatic analysis of two-dimensional(2D) projections of this 6D experiment with the use of GAPRO (Hilleretal., 2005) provides a high-precision 6D peak list, which permits automated sequential assignments of proteins with the assignment software GARANT (Bartels et al., 1997). The procedure was applied to two proteins, the 63-residue 434-repressor(1-3) and the 115-residue TM1290. For both proteins, complete sequential assignments for all NMR-observable backbone resonances were obtained, and the polypeptide segments thus identified could be unambiguously located in the amino acid sequence. These results demonstrate that APSY-NMR spectroscopy in combination with a suitable assignment algorithm can provide fully automated sequence-specific backbone assignments of small proteins.