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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Kay, L.E. Solution NMR spectroscopy of supra-molecular systems, why bother? A methyl-TROSY view. J. Magn. Reson. 210, 159–170 (2011).
Fawzi, N.L., Ying, J., Ghirlando, R., Torchia, D.A. & Clore, G.M. Atomic-resolution dynamics on the surface of amyloid-β protofibrils probed by solution NMR. Nature 480, 268–272 (2011).
Fawzi, N.L., Ying, J., Torchia, D.A. & Clore, G.M. Kinetics of amyloid β monomer-to-oligomer exchange by NMR relaxation. J. Am. Chem. Soc. 132, 9948–9951 (2010).
Clore, G.M. & Iwahara, J. Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low-population states of biological macromolecules and their complexes. Chem. Rev. 109, 4108–4139 (2009).
Ishima, R. & Torchia, D.A. Accuracy of optimized chemical-exchange parameters derived by fitting CPMG R2 dispersion profiles when R20a≠R20b. J. Biomol. NMR 34, 209–219 (2006).
Roder, H., Elove, G.A. & Englander, S.W. Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR. Nature 335, 700–704 (1988).
Carulla, N., Zhou, M., Giralt, E., Robinson, C.V. & Dobson, C.M. Structure and intermolecular dynamics of aggregates populated during amyloid fibril formation studied by hydrogen/deuterium exchange. Acc. Chem. Res. 43, 1072–1079 (2010).
Clore, G.M. & Gronenborn, A.M. Theory of the time-dependent transferred nuclear Overhauser effect—applications to structural analysis of ligand protein complexes in solution. J. Magn. Reson. 53, 423–442 (1983).
Post, C.B. Exchange-transferred NOE spectroscopy and bound ligand structure determination. Curr. Opin. Struct. Biol. 13, 581–588 (2003).
Bodner, C.R., Dobson, C.M. & Bax, A. Multiple tight phospholipid-binding modes of α-synuclein revealed by solution NMR spectroscopy. J. Mol. Biol. 390, 775–790 (2009).
Cavanagh, J., Fairbrother, W.J., Palmer, A.G. III, Rance, M. & Skelton, N.J. Protein NMR spectroscopy: Principles and Practice, 2nd edn. (Academic Press, 2007).
Iwahara, J., Jung, Y.S. & Clore, G.M. Heteronuclear NMR spectroscopy for lysine NH3 groups in proteins: unique effect of water exchange on 15N transverse relaxation. J. Am. Chem. Soc. 129, 2971–2980 (2007).
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.).
Author information
Authors and Affiliations
Contributions
All the authors contributed extensively to the work described in this paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2012.077
This article is cited by
-
The role of NMR in leveraging dynamics and entropy in drug design
Journal of Biomolecular NMR (2020)
-
Molecular interactions underlying liquid−liquid phase separation of the FUS low-complexity domain
Nature Structural & Molecular Biology (2019)
-
Exchange saturation transfer and associated NMR techniques for studies of protein interactions involving high-molecular-weight systems
Journal of Biomolecular NMR (2019)
-
Tuning a timing device that regulates lateral root development in rice
Journal of Biomolecular NMR (2019)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.