Solution NMR Experiment for Measurement of ¹⁵N-¹H Residual Dipolar Couplings in Large Proteins and Supramolecular Complexes

NMR residual dipolar couplings (RDCs) are exquisite probes of protein structure and dynamics. A new solution NMR experiment named 2D SE2 J-TROSY is presented to measure N-H RDCs for proteins and supramolecular complexes in excess of 200 kDa. This enables validation and refinement of their X-ray crystal and solution NMR structures and the characterization of structural and dynamic changes occurring upon complex formation. Accurate N-H RDCs were measured at 750 MHz ¹H resonance frequency for 11-mer 93 kDa ²H,¹⁵N-labeled Trp RNA-binding attenuator protein tumbling with a correlation time τ_c of 120 ns. This is about twice as long as that for the most slowly tumbling system, for which N-H RDCs could be measured, so far, and corresponds to molecular weights of ∼200 kDa at 25 °C. Furthermore, due to the robustness of SE2 J-TROSY with respect to residual ¹H density from exchangeable protons, increased sensitivity at ¹H resonance frequencies around 1 GHz promises to enable N-H RDC measurement for even larger systems.