A Suite of Triple Resonance NMR Experiments for the Backbone Assignment of ¹⁵N, ¹³C, ²H Labeled Proteins with High Sensitivity

A suite of NMR pulse schemes is presented for the assignment of HN, ¹⁵N, ¹³C^α, and ¹³C^β resonances in ¹⁵N, ¹³C, ²H labeled proteins. The experiments exploit the line narrowing of the ¹³C spins caused by the substitution of deuterons for bound protons and are therefore well suited for application to molecules with masses on the order of 30-40 kDa where standard triple resonance experiments may fail completely or provide spectra of poor sensitivity. The methods are demonstrated on a 37 kDa ternary complex of a ¹⁵N, ¹³C, and ~70% ²H labeled sample of tip-repressor, the corepressor 5-methyltryptophan, and a 20 basepair trp-operator DNA fragment. All of the experiments in the family presented provide well over 95% of the expected intra-and/or inter-residue correlations. Carbon relaxation measurements of the complex indicate that the use of deuteration increases the average ¹³C^α T₂ value from 16.5 to 130 ms.