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

Abstract

The NMR assignment of ¹³C, ¹⁵N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below ∼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 (¹H^N), 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.