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, and/or covalent conjugation with a fluorescent or paramagnetic tag), and provide a comprehensive characterization of conformational equilibria returning structural, kinetic, and thermodynamic information on exchange processes. In order for a CPMG experiment to report on a conformational equilibrium, two conditions must apply: (i) the observed NMR spins must possess different chemical shifts in the states undergoing conformational exchange (microstates) and (ii) the exchange has to occur at a time scale ranging from ~50 µs to ~10 ms. Under these conditions, the observed transverse relaxation rate () is the sum of the intrinsic R2 (the R2 measured in the absence of µs-ms dynamics, ) and the exchange contribution to the transverse relaxation (Rex). The Rex contribution to R2obs can be progressively quenched by reducing the spacing between the 180° pulses constituting the CPMG block of the pulse sequence, and the resulting RD curves can be modeled using the Bloch-McConnell theory to obtain the chemical shift difference among microstates, the fractional population of each microstate, and the rates of exchange among microstates (Figure 1)1,2,3.
Several different pulse sequences and analysis protocols have been reported in the literature for 15N CPMG experiments. Herein, the protocol implemented in the laboratory is described. In particular, the crucial steps for preparation of the NMR sample, set up and acquisition of the NMR experiments, and processing and analysis of the NMR data will be introduced (Figure 2). To facilitate transfer of the protocol to other laboratories, the pulse program, processing and analysis scripts, and one example dataset are provided as Supplemental Files and are available for download at (https://group.chem.iastate.edu/Venditti/downloads.html). The provided pulse sequence incorporates a four-step phase cycle in the CPMG block for suppression of offset-dependent artifacts6 and it is coded for acquisition of several interleaved experiments. These interleaved experiments have an identical relaxation period but different numbers of refocusing pulses in order to achieve different CPMG fields7. It is also important to notice that the described pulse program measures the 15N R2 of the TROSY component of the NMR signal8. Overall, the protocol has been successfully applied for the characterization of conformational exchange in medium and large-sized proteins4,5,9,10. For smaller systems (<20 kDa), the use of an Heteronuclear Single Quantum Coherence (HSQC)-based pulse sequence11,12 is advisable.
1. Preparation of the NMR sample
2. First time set-up of the NMR experiment
3. Routine set-up of the NMR experiment
4. Processing and analysis of the NMR data
5. Fitting RD curves
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 regions undergoing conformational exchange on the µs-ms time scale (Figure 3C). Modeling of the RD curves using the Carver-Richards equation returns thermodynamic and kinetic parameters on the exchange process, such as the fractional populations of the states in equilibrium and the rate of exchange among these states (Figure 1, Figure 3D). The temperature dependence of these thermodynamic and kinetic parameters (obtained by acquiring RD experiments at multiple experimental temperatures) can be modeled using the van't Hoff and Eyring equations, respectively, to obtain detailed information on the energetics of the conformational exchange (Figure 3E)9,10.
Figure 1: Overview of the CPMG RD experiment. (A) Schematic view of the CPMG block used for acquisition of RD data. The 180° pulses are shown as black rectangles. The operator indicates the magnetization that enters and exits the CPMG block. The CPMG field is determined by the spacing between subsequent refocusing pulses (2τ). (B) In a two-time point measurement, the relaxation delay (during which the CPMG block is applied) is kept constant and the CPMG field is varied by varying the values of n (the number of times the CPMG block is applied during the relaxation delay period) and τ. (C) Schematic representation of a two-site equilibrium between conformations A and B. The exchange rate constant (kex) is the sum of the forward and reverse rate constants kab and kba, respectively). pa and pb (= 1 – pa) are the fractional populations of species A and B, respectively. Δωab is the chemical shift difference between conformations A and B. (D) Simulated RD curves for exchange processes that are in the range accessible by CPMG (top left), too fast to be detected by CPMG (top right), too slow to be detected by CPMG (bottom right), and with a Δωab too small to be detected by CPMG (bottom left). The pb value was set to 3%. Please click here to view a larger version of this figure.
Figure 2: Main pipeline for acquisition and analysis of RD data. Schematic representation of the workflow described in the present protocol. Please click here to view a larger version of this figure.
Figure 3: µs-ms dynamics of EIC by CPMG RD experiments. (A) Example 15N RD profiles measured at 40 °C and 800 MHz for EIC using the protocol described here. The estimation of Rex is shown in red. (B) Rex values are plotted versus the residue index and (C) on the X-ray structure of the enzyme to identify residues undergoing conformational exchange. (D) NMR signals with Rex larger than the error are modeled (using the script provided in Supplemental Files) to obtain the kinetics (kab and kba) and thermodynamics (pb) of the equilibrium. (E) Acquisition of RD data at multiple temperatures returns information on the energetics of the conformational change. Please click here to view a larger version of this figure.
Supplemental Files. Please click here to download this File.
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 recommended3,8,11,15,16.
When preparing the NMR sample, it is extremely important to consider that the presence of any minor (<1% populated) state in exchange with the major, NMR visible species on the µs-ms timescale will generate detectable RD profiles1. Therefore, it is recommended to use a highly purified protein stock (>90% pure) to avoid the presence of contaminants that could form transient complexes with the system under investigation. In addition, if investigating conformational dynamics in protein-ligand complexes, it is important to use saturating concentrations of ligand in order to avoid the presence of spurious RD profiles originating by the kinetics of ligand binding.
When working with high molecular weight systems (>20 kDa), it is also advisable (although not necessary) to use perdeuterated protein samples and the TROSY pulse sequence presented with the present protocol to reduce the transverse relaxation rate and maximize the relaxation period (d30 in our pulse program). Indeed, as the lowest obtainable νcpmg. = 4/d30, using a long relaxation period allows acquisition of R2 data at small νcpmg, where the exchange contribution to the R2 is at its maximum. In this respect, it is also important to mention that a pulse sequence similar to the one provided with the present protocol is present in the standard pulse sequence portfolio of the spectrometer (file name: trhncorexf3gp). The main difference between the two files is that the standard sequence is based on a TROSY-HNCO experiment, while our experiment is based on a TROSY-HSQC experiment and does not require 13C labeling of the sample.
Another issue to consider carefully is the acquisition temperature. Indeed, as the RD data are usually measured at multiple static fields, it is crucial that the acquisition temperature is consistent among all spectrometers used. Therefore, it is highly recommended to check the temperature calibration before setting up the experiment.
For what concerns the analysis of the RD curves, it is important to stress out that the procedures and fitting scripts presented here make use of the Carver-Richards equations. While this is the most common procedure applied in the literature for quantitative modeling of the RD data, the Carver Richards equations incorporate a number of approximations and are limited to the two-site exchange case17. If a particular dataset requires the more rigorous Bloch-McConnell matrices for data modeling, the fitting procedure should be modified accordingly18,19,20. In the protocol above, a few freely available software packages are listed that perform data modeling using the Bloch-McConnell theory.
Finally, it should be noted that, although our manuscript focuses solely on the application of 15N RD data for the investigation of protein conformational dynamics, several other experiments were described in the literature to measure RD curves on different nuclei and other biological and non-biological molecular systems18,19,21,22,23. In particular the use of different nuclei is extremely important, as it allows a more dense sampling of the protein structure and provides information on side-chain dynamics that are largely disregarded by the 15N-based experiment presented in this protocol5,21,23.
The authors have nothing to disclose.
This work was supported by funds from NIGMS R35GM133488 and from the Roy J. Carver Charitable Trust to V.V.
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 |