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Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy

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Abstract

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.

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Figure 1: Summary of the kinetic models available in the DESTfit program.
Figure 2: Screenshots of figures output by the DESTfit software.

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Acknowledgements

We thank D. Libich for helpful comments. This work was supported by the intramural program of NIDDK/NIH and the AIDS Targeted Antiviral Program of the NIH Director (to G.M.C.).

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All the authors contributed extensively to the work described in this paper.

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Correspondence to G Marius Clore.

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Fawzi, N., Ying, J., Torchia, D. et al. Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy. Nat Protoc 7, 1523–1533 (2012). https://doi.org/10.1038/nprot.2012.077

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