Molecular complexes often sample conformational states that direct them to specific functions. These states can be difficult to observe through traditional biophysical approaches but they can be studied using a variety of different NMR spin relaxation experiments. However, these applications, when focused on moderate to high molecular weight proteins, are complicated by fast relaxing signals that negatively affect the sensitivity and resolution of spectra. Here a methyl 1 H CPMG-based experiment for studies of excited conformational states of protein machines is described that exploits a TROSY-effect to increase signal-to-noise. Complexities from the multiplicity of methyl 1 H transitions are addressed to generate a robust pulse scheme that is applied to a 320 kDa homeostasis protein, p97.
Keywords: CPMG; degenerate transitions; invisible excited states; methyl NMR; molecular machines.
© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.