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

J Am Chem Soc. 2015 Sep 9;137(35):11242-5. doi: 10.1021/jacs.5b07010. Epub 2015 Aug 27.

Abstract

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 (1)H resonance frequency for 11-mer 93 kDa (2)H,(15)N-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 (1)H density from exchangeable protons, increased sensitivity at (1)H resonance frequencies around 1 GHz promises to enable N-H RDC measurement for even larger systems.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / chemistry
  • Magnetic Resonance Spectroscopy / methods*
  • Models, Molecular
  • Molecular Weight
  • Protein Conformation
  • Proteins / chemistry*
  • RNA-Binding Proteins / chemistry
  • Solutions
  • Transcription Factors / chemistry

Substances

  • Bacterial Proteins
  • MtrB protein, Bacteria
  • Proteins
  • RNA-Binding Proteins
  • Solutions
  • Transcription Factors