High-throughput backbone resonance assignment of small 13C, 15N-labeled proteins by a triple-resonance experiment with four sequential connectivity pathways using chemical shift-dependent, apparent [formula omitted]( 1H, 13C): HNCACB codedHAHB

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The proposed three-dimensional triple-resonance experiment HNCACB codedHAHB correlates sequential 15N, 1H moieties via the chemical shifts of 13C α, 13C β, 1H α, and 1H β. The four sequential correlation pathways are achieved by the incorporation of the concept of chemical shift-coding [J. Biomol. NMR 25 (2003) 281] to the TROSY-HNCACB experiment. The monitored 1H α and 1H β chemical shifts are then coded in the line shape of the cross-peaks of 13C α, 13C β along the 13C dimension through an apparent residual scalar coupling, the size of which depends on the attached hydrogen chemical shift. The information of four sequential correlation pathways enables a rapid backbone assignment. The HNCACB codedHAHB experiment was applied to ∼85% labeled 13C, 15N-labeled amino-terminal fragment of Vaccinia virus DNA topoisomerase I comprising residues 1–77. After one day of measurement on a Bruker Avance 700 MHz spectrometer and 8 h of manual analysis of the spectrum 93% of the backbone assignment was achieved.