We present the protocol for the measurement and analysis of dark-state exchange saturation transfer (DEST), a novel solution NMR method for characterizing, at atomic resolution, the interaction between an NMR-'visible' free species and an NMR-'invisible' species transiently bound to a very high-molecular-weight (>1 MDa) macromolecular entity. The reduced rate of reorientational motion in the bound state that precludes characterization by traditional NMR methods permits the observation of DEST. 15N-DEST profiles are measured on a sample comprising the dark state in exchange with an NMR-visible species; in addition, the difference (ΔR2) in 15N transverse relaxation rates between this sample and a control sample comprising only the NMR-visible species is also obtained. The 15N-DEST and ΔR2 data for all residues are then fitted simultaneously to the McConnell equations for various exchange models describing the residue-specific dynamics in the bound state(s) and the interconversion rate constants. Although the length of the experiments depends strongly on sample conditions, approximately 1 week of NMR spectrometer time was sufficient for full characterization of samples of amyloid-β (Aβ) at concentrations of ∼100 μM. © 2012 Nature America, Inc. All rights reserved.
CITATION STYLE
Fawzi, N. L., Ying, J., Torchia, D. A., & Clore, G. M. (2012). Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy. Nature Protocols, 7(8), 1523–1533. https://doi.org/10.1038/nprot.2012.077
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