A new 3D HN(CA)HA experiment for obtaining fingerprint H^N-H^α cross peaks in¹⁵N- and¹³C-labeled proteins

Summary

A new 3D¹H−¹⁵N−¹³C triple resonance experiment is presented that provides in-phase absorptive cross peaks between amide protons and α-protons of the same and the sequentially preceding residue. The experiment yields similar connectivities as those described previously by Montelione and Wagner (1990a) [J. Magn. Reson.,87, 183–188] and Kay et al. (1991) [J. Magn. Reson.,91, 84–92]. However, the pulse sequence was designed to minimize the time that transverse coherence of the¹³C^α nucleus is present, since this nucleus has the shortest transverse relaxation time of all the nuclei involved in these experiments. This is achieved by using a coherence transfer pathway from¹H^N to¹⁵N,¹³C^α,¹H^α and back to the¹H^N. In the sequence described, transverse¹³C^α coherence is present only for a length of ca. I¹J(C^α-H^α). This reduces loss of signal due to transverse relaxation. We tested the technique on uniformly¹⁵N- and¹³C-enriched T4 lysozyme.