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

Summary

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.

Abstract

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.

Introduction

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 spectroscopy^1,2,3,4,5. 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,....

Protocol

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 NMR buffer.
3. Transfer the NMR sample into the NMR tube.
   NOTE: The concentration of the NMR sample needs to be carefully optimized in ord....

Results

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 R_ex on the 3D structure of the protein under investigation, it is possible to identify the structural reg.......

Discussion

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

Materials

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