A new 3D HN(CA)HA experiment for obtaining fingerprint H N -H α cross peaks in 15 N- and 13 C-labeled proteins

Abstract

A new 3D 1 H− 15 N− 13 C 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 the 13 C 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 from 1 H N to 15 N, 13 C α , 1 H α and back to the 1 H N . In the sequence described, transverse 13 C α coherence is present only for a length of ca. I 1 J(C α -H α ). This reduces loss of signal due to transverse relaxation. We tested the technique on uniformly 15 N- and 13 C-enriched T4 lysozyme.