TROSY in triple-resonance experiments: New perspectives for sequential NMR assignment of large proteins

TLDR

NMR assignment of 13C,15N‑labeled proteins using triple‑resonance experiments is limited to ~25 kDa because of rapid transverse relaxation, but deuterium labeling of amide protons, nitrogens, and carbons has previously extended this size limit. The authors present pulse schemes and spectra for [15N,1H]‑TROSY‑HNCA and HNCO experiments on deuterated and protonated proteins, and theoretically show that TROSY primarily reduces 15N transverse relaxation, yielding predicted sensitivity gains. Implementing [15N,1H]‑TROSY in triple‑resonance experiments yields several‑fold sensitivity gains for 2H/13C/15N‑labeled proteins and about two‑fold gains for 13C/15N‑labeled proteins, matching theoretical predictions.

Abstract

The NMR assignment of 13C, 15N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below approximately 25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, this size limit has been previously extended by additional labeling with deuterium (2H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([15N,1H]-TROSY) into triple resonance experiments results in several-fold improved sensitivity for 2H/13C/15N-labeled proteins and approximately twofold sensitivity gain for 13C/15N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [15N, 1H]-TROSY-HNCA and [15N, 1H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of 15N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.