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.