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

Published: April 19th, 2021



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 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 NMR data processing
SPARKY University of California, San Francisco Analysis of the NMR data
Tospin 3.2 (or newer) Bruker acquisition software

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