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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Biochemistry

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the µs-ms Timescale

Published: April 19th, 2021

DOI:

10.3791/62395

1Department of Chemistry, Iowa State University, 2Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University

Here, a detailed description of the protocol implemented in the laboratory for acquisition and analysis of 15N relaxation dispersion profiles by solution NMR spectroscopy is provided.

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important biological processes. Understanding how the balance between structure and dynamics governs biological function is a new frontier in modern structural biology and has ignited several technical and methodological developments. Among these, CPMG relaxation dispersion solution NMR methods provide unique, atomic-resolution information on the structure, kinetics, and thermodynamics of protein conformational equilibria occurring on the µs-ms timescale. Here, the study presents detailed protocols for acquisition and analysis of a 15N relaxation dispersion experiment. As an example, the pipeline for the analysis of the µs-ms dynamics in the C-terminal domain of bacteria Enzyme I is shown.

Carr-Purcell Meiboom-Gill (CPMG) relaxation dispersion (RD) experiments are used on a routine base to characterize conformational equilibria occurring on the µs-ms timescale by solution NMR spectroscopy1,2,3,4,5. Compared to other methods for investigation of conformational dynamics, CPMG techniques are relatively easy to implement on modern NMR spectrometers, do not require specialized sample preparation steps (i.e., crystallization, sample freezing or alignment,....

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1. Preparation of the NMR sample

  1. Express and purify a 2H,15N-labled sample of the protein of interest.
    NOTE: While a 15N-labeled protein sample can be used for acquisition of the CPMG RD experiment, perdeuteration (where possible) dramatically increases the quality of the obtained data. Protocols for the production of perdeuterated proteins are available in the literature13.
  2. Buffer exchange the purified protein sample into a degassed NM.......

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The protocol described here results in acquisition of RD profiles for each peak in the 1H-15N TROSY spectrum (Figure 3A). From the acquired RD profiles, it is possible to estimate the exchange contribution to the 15N transverse relaxation of each backbone amide group (Figure 3A,3B). By plotting the Rex on the 3D structure of the protein under investigation, it is possible to identify the structural reg.......

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This manuscript describes the protocol implemented in the laboratory for acquisition and analysis of 15N RD data on proteins. In particular, the crucial steps for preparation of the NMR sample, measurement of the NMR data, and analysis of the RD profiles are covered. Below some important aspects regarding the acquisition and analysis of RD experiments are discussed. However, for a more in-depth description of the experiment and data analysis, careful studying of the original literature is highly recommended

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This work was supported by funds from NIGMS R35GM133488 and from the Roy J. Carver Charitable Trust to V.V.

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Name Company Catalog Number Comments
Cryoprobe Bruker 5mm TCI 800 H-C/N-D cryoprobe Improve sensitivity
Deuterium Oxide Sigma Aldrich 756822-1 >99.8% pure, utilised in preparing NMR samples and deuterated cultures
Hand driven centrifuge United Scientific supply CENTFG1 Used to remove any air bubbles or residual liquid stuck on the walls of NMR tube.
High Field NMR spectrometer Bruker Bruker Avance II 600, Bruker Avance 800 acquisition of the NMR data
MATLAB MathWorks https://www.mathworks.com/products/get-matlab.html Modeling of the NMR data
NMR pasteur Pipette Corning Incorporation 7095D-NMR Pyrex glass pastuer pipette to transfer liquid sample in NMR tube
NMR tube Willmad Precision 535-PP-7 5mm thin wall 7'' cylinderical glass tube
NMRPipe Institute of Biosciences and Biotechnology research https://www.ibbr.umd.edu/nmrpipe/install.html NMR data processing
SPARKY University of California, San Francisco https://www.cgl.ucsf.edu/home/sparky/ Analysis of the NMR data
Tospin 3.2 (or newer) Bruker https://www.bruker.com/protected/en/services/software-downloads/nmr/pc/pc-topspin.html acquisition software

  1. Anthis, N. J., Clore, G. M. Visualizing transient dark states by NMR spectroscopy. Quarterly Reviews of Biophysics. 48 (1), 35-116 (2015).
  2. Lisi, G. P., Loria, J. P. Solution NMR spectroscopy for the study of enzyme allostery. Chemical Reviews. 116 (11), 6323-6369 (2016).
  3. Mittermaier, A., Kay, L. E. New tools provide new insights in NMR studies of protein dynamics. Science. 312 (5771), 224-228 (2006).
  4. Venditti, V., Clore, G. M. Conformational selection and substrate binding regulate the monomer/dimer equilibrium of the C-terminal domain of Escherichia coli enzyme I. Journal of Biological Chemistry. 287 (32), 26989-26998 (2012).
  5. Venditti, V., et al. Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I. Nature Communications. 6, 5960 (2015).
  6. Yip, G. N., Zuiderweg, E. R. A phase cycle scheme that significantly suppresses offset-dependent artifacts in the R2-CPMG 15N relaxation experiment. Journal of Magnetic Resonance. 171 (1), 25-36 (2004).
  7. Mulder, F. A., Skrynnikov, N. R., Hon, B., Dahlquist, F. W., Kay, L. E. Measurement of slow (micros-ms) time scale dynamics in protein side chains by (15)N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme. Journal of the American Chemical Society. 123 (5), 967-975 (2001).
  8. Loria, J. P., Rance, M., Palmer, A. G. A TROSY CPMG sequence for characterizing chemical exchange in large proteins. Journal of Biomolecular NMR. 15 (2), 151-155 (1999).
  9. Dotas, R. R., et al. Hybrid thermophilic/mesophilic enzymes reveal a role for conformational disorder in regulation of bacterial Enzyme I. Journal of Molecular Biology. 432 (16), 4481-4498 (2020).
  10. Purslow, J. A., et al. Active site breathing of human Alkbh5 revealed by solution NMR and accelerated molecular dynamics. Biophysical Journal. 115, 1895-1905 (2018).
  11. Loria, J. P., Rance, M., Palmer, A. G. A relaxation-compensated Carr−Purcell−Meiboom−Gill sequence for characterizing chemical exchange by NMR Spectroscopy. Journal of the American Chemical Society. 121 (10), 2331-2332 (1999).
  12. Hansen, D. F., Vallurupalli, P., Kay, L. E. An improved 15N relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins. Journal of Physical Chemistry B. 112 (19), 5898-5904 (2008).
  13. Tugarinov, V., Kanelis, V., Kay, L. E. Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nature Protocols. 1 (2), 749-754 (2006).
  14. Niklasson, M., et al. Comprehensive analysis of NMR data using advanced line shape fitting. Journal of Biomolecular NMR. 69, 93-99 (2017).
  15. Palmer, A. G., Kroenke, C. D., Loria, J. P. Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Methods in Enzymology. 339, 204-238 (2001).
  16. Tollinger, M., Skrynnikov, N. R., Mulder, F. A., Forman-Kay, J. D., Kay, L. E. Slow dynamics in folded and unfolded states of an SH3 domain. Journal of the American Chemical Society. 123, 11341-11352 (2001).
  17. Carver, J. P., Richards, R. E. A general two-site solution for the chemical exchange produced dependence of T2 upon the Carr-Purcell pulse separation. Journal of Magnetic Resonance. 6 (1), 89-105 (1972).
  18. Egner, T. K., et al. Surface Contrast' NMR Reveals Non-innocent Role of Support in Pd/CeO2 Catalyzed Phenol Hydrogenation. ChemCatChem. 12 (6), 4160-4166 (2020).
  19. Egner, T. K., Naik, P., Nelson, N. C., Slowing, I. I., Venditti, V. Mechanistic Insight into Nanoparticle Surface Adsorption by Solution NMR Spectroscopy in an Aqueous Gel. Angewandte Chemie (International Edition in English). 56, 9802-9806 (2017).
  20. Tugarinov, V., Libich, D. S., Meyer, V., Roche, J., Clore, G. M. The energetics of a three-state protein folding system probed by high-pressure relaxation dispersion NMR spectroscopy. Angewandte Chemie (International Edition in English). 54, 11157-11161 (2015).
  21. Korzhnev, D. M., Kloiber, K., Kanelis, V., Tugarinov, V., Kay, L. E. Probing slow dynamics in high molecular weight proteins by methyl-TROSY NMR spectroscopy: application to a 723-residue enzyme. Journal of the American Chemical Society. 126 (12), 3964-3973 (2004).
  22. Mayzel, M., Ahlner, A., Lundstrom, P., Orekhov, V. Y. Measurement of protein backbone (13)CO and (15)N relaxation dispersion at high resolution. Journal of Biomolecular NMR. 69, 1-12 (2017).
  23. Pritchard, R. B., Hansen, D. F. Characterising side chains in large proteins by protonless (13)C-detected NMR spectroscopy. Nature Communications. 10, 1747 (2019).

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