Assignment of Backbone Resonances for Larger Proteins Using the 13C−1H Coherence of a 1Hα-, 2H-, 13C-, and 15N-Labeled Sample

A suite of triple resonance NMR experiments for the assignment of backbone resonances of a larger protein using selectively 1 H α labeling to a sample uniformly labeled with 13 C, 15 N, and 2 H is described. The relaxation time of 1 H α − 13 C α zero/double quantum coherence was more than 4 times as long as that of 13 C α single quantum coherence. Three-dimensional HACAN, HACACB, HACACO, and HACA(N)CO experiments were newly designed to utilize selectively labeled 1 H α nuclei. HACAN provides intraresidue and sequential connectivities through amide 15 N spins. HACACO and HACA(N)CO provide intraresidue and sequential connectivities through 13 CO spins. HACACB provides connectivity to 13 C β, giving the type of amino acid. Long-life 1 H α − 13 C α zero/double quantum coherence provides high sensitivity in these NMR experiments. Except for a few amino acid type-specific problems, all sequential connectivities were obtained for a test sample of a 98 amino acid protein at 10 °C, which rotationally diffuses with a correlation time of 17 ns, corresponding to an over 30 kDa protein at 30−40 °C. Zero/double quantum based triple resonance experiments and 1 H α selective labeling provide a new approach for NMR studies on larger proteins.