ja5b07010_si_001.pdf (1.01 MB)
Solution NMR Experiment for Measurement of 15N–1H Residual Dipolar Couplings in Large Proteins and Supramolecular Complexes
journal contribution
posted on 2015-09-09, 00:00 authored by Alexander Eletsky, Surya
V.S.R.K. Pulavarti, Victor Beaumont, Paul Gollnick, Thomas SzyperskiNMR
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 τc 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.