We thank Melinda Jaspert and Kevin Bochinsky for the helpful discussions. N.L. thanks the German Science Foundation for funding through the Heisenberg Program (DFG grant number 433700474). This work is further supported by the project &#34;Virological and immunological determinants of COVID-19 pathogenesis - lessons to get prepared for future pandemics (KA1-Co-02 &#34;COVIPA&#34;), a grant from the Helmholtz Association&#39;s Initiative and Networking Fund. We acknowledge generous access to the J&#252;lich-D&#252;sseldorf Biomolecular NMR Center, jointly run by Forschungszentrum J&#252;lich and Heinrich Heine University D&#252;sseldorf (HHU).

1. NMR sample preparation
NOTE: Isotope labeling of the proteins is performed for higher-dimensional NMR and advanced NMR experiments. When protein expression in E. coli and protein purification had been established using rich media (e.g., Luria-Bertani [LB] or 2x yeast extract tryptone medium [2YT]) with a yield of several milligrams per liter, preparing an isotopically labeled NMR sample is usually relatively straightforward.
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	<li>For isotope labeling, use M9...

Protocol for NMR &lt;sup&gt;15&lt;/sup&gt;N Relaxation and hetNOE Experiments in Protein Dynamics Study

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This protocol described the setup of NMR 15N relaxation experiments by Lakomek et al.69 and Stief et al.70. We focused on NMR pulse sequences using a sensitivity-enhanced HSQC detection scheme. The 15N R1 and R1&#961; experiments are implemented as described in detail by Stief et al.70, and the hetNOE experiment is described by Lakomek et al.69.
When setti...

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			<td>Bruker 600 MHz AVANCE III HD spectrometer&#160;</td>
			<td>Bruker</td>
			<td>https://www.bruker.com/en/products-and-solutions/mr/nmr/avance-nmr-spectrometer.html</td>
			<td>NMR experiments conducted&#160;</td>
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nmr 15n relaxation experiments for the investigation of picosecond to nanoseconds structural dynamics of proteins

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on 15N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard 15N relaxation measurements overview a protein&#39;s internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR 15N R1, R2, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.

Author Spotlight: Exploring Intrinsically Disordered Protein Dynamics Through NMR Relaxation Experiments

NMR 15N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins

We investigate the structural dynamics of proteins by NMR spectroscopy to understand the molecular function better. NMR stands for Nuclear Magnetic Resonance Spectroscopy. It reduces effect that the nuclear spins of hydrogen atoms can transition between two energetically different states, and an auto magnetic field.We can use NMR spectroscopy to study the structural dynamics of intrinsically disordered proteins. These proteins are highly flexible, constantly changing shape, and frequently only fold into a stable structure when they interact with other molecules. We use NMR relaxation experiments to study how the protein backbone moves over very short time scales, ranging from picoseconds to nanoseconds, while focusing on each specific residue along the protein chain.This protocol will guide you step-by-step through setting up NMR relaxation experiments on an NMR spectrometer aimed at scientists with basic NMR knowledge, it ensures a fast, accurate setup, and introduces key concepts in protein backbone dynamics in an accessible way. This protocol makes NMR relaxation pulse sequences easily accessible to scientists studying protein dynamics. NMR relaxation experiments provide residue specific dynamic insights.Using this experimental approach, you can better understand backbone dynamics in proteins, the impact of protein-protein interaction on structured dynamics, and structural changes in the response to environmental changes.

The following shows some exemplary NMR relaxation data recorded on the vesicular SNARE protein Synaptobrevin-2 (1-96), frequently called VAMP2 (vesicle-associated protein 2). For recording the NMR data, we used a 171 &#181;M 15N Synaptobrevin-2 (1-96) sample (dubbed Syb-2 in the following) in 50 mM MES (pH 6.0) buffer containing 150 mM NaCl, 0.1 mM TCEP, and 1 mM EDTA. All experimental data was recorded at 278.15 K using a 250 &#181;L volume filled in a 3 mm NMR sample tube. Experiments were performed at a Bru...

Nuclear magnetic resonance (NMR) spectroscopy can characterize structural protein dynamics in a residue-specific manner. We provide a hands-on protocol for recording NMR 15N R1 and R2 relaxation and {1H}-15N heteronuclear Overhauser effect (hetNOE) experiments, sensitive to the picoseconds to nanoseconds timescale.

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide ...

nmr-15n-relaxation-experiments-for-investigation-picosecond-to

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JoVE Journal

Biochemistry

Running the NMR Relaxation Experiments on the Spectrometer

To begin, download the NMR pulse programs to the directory on the computer operating the NMR spectrometer and adjust the TopSpin version. Download the NMR shaped pulses to the directory and verify that the TopSpin version is updated accordingly. After determining the spectral widths and appropriate acquisition times, type edc in the TopSpin software to a new directory to copy the HSQC experiment.To open the pulse sequence parameters type ased, click on the three dots next to the PULPROG parameter to change the pulse program to nitrogen-15 R1 rho experiment. Double-click on the pulse program, click on Set PULPROG to the dataset and confirm with OK.Type ased to open the pulse sequence parameters. Enter all missing gradient strengths and gradient lengths as given in the pulse sequence.Next, enter for the loop counter L3 half of the nitrogen-15 TD entry in the acquisition parameter window. Enter for the loop counter L6 the number of recorded relaxation data points for fitting the relaxation curve. Set the adiabatic TanhTan_half.nl pulse as the shape of the nitrogen-15 SP8 pulse. Then set the adiabatic TanhTan_half2nd. nl pulse as the shape of the nitrogen-15 SP9 pulse.Ensure the pulse lengths are sufficiently long for adiabaticity with P8 set to 3, 000 microseconds. Then set the interscan recovery delay, D1, to at least two seconds or longer. Set the dummy scans to at least 64.As a starting point set the number of scans to four and use multiples of four if the signal-to-noise ratio is too low. Set the O1 to the calibrated carrier frequency, O2P to 176 parts per million, and copy the O3P from the proton nitrogen-15 HSQC experiment. Now, set the pulse length P7 to the 90-degree pulse length calibrated earlier.Then copy the pulse power level of the 90-degree pulse to PLW3 and PLW7. Afterward, set the pulse lengths P1 and P19 to the 90-degree proton pulse length. Set the number of increments in the indirect dimension, TD equals L3 by two by L6.Next, set the shaped pulse SP5 to an I-BURP2 shape and the pulse length P15 to 2, 000 microseconds.Then open the Shape Tool display by clicking the E next to the I-BURP2 shaped pulse in the TopSpin pulse sequence parameter window. To simulate the shaped pulse click the Start NMR Simulation button. Check the Shaped pulse length and the rotation angle in the simulation window and click on Start NMR SIM to proceed.Check the excitation range in the simulation and select the appropriate I-BURP2 pulse length to cover the proton spectral dispersion while avoiding excitation of the water. Set P15 to the shaped pulse length from the simulation window with the best I-BURP2 pulse. Now, set SPOFFS5 to adjust the carrier frequency of the I-BURP2 pulse, shifting the excitation range left or right to avoid water magnetization disturbance.Then open the Bruker Shape Tool and click Start NMR Simulation to determine the appropriate power level of the shaped pulse. Set the I-BURP2 pulse length to the shaped pulse length, and note the soft rectangular 90-degree proton pulse length shown in the simulation window. Type calcpowlev to calculate the power level difference in decibels between the hard 90-degree proton pulse and the soft rectangular 90-degree proton pulse.Copy the power level of the hard 90-degree proton pulse to SPW5 and adjust by adding the memorized difference in decibels. To determine the power level of the spin-lock calculate the corresponding 90-degree nitrogen-15 pulse length. Use calcpowlev to calculate the power difference in decibels between the spin-lock power and the hard 90-degree nitrogen-15 pulse.Copy the power level of the hard 90-degree nitrogen-15 pulse to PLW7 and adjust the spin-lock power level PLW8 by adding the calculated power difference. Copy the power level of the spin-lock PLW8 to the power levels SPW8 and SPW9. To determine the power level of the nitrogen-15 decoupling use calcpowlev to calculate the power difference between the power of the 90-degree nitrogen-15 decoupling pulse and the 90-degree nitrogen-15 hard pulse.Copy the power level of the 90-degree nitrogen-15 hard pulse PLW7 to the decoupling power level PLW31 and adjust by adding the calculated power difference in decibels. For temperature compensation include line define TEMP_ in the pulse program. Set P18 to the maximum duration of the spin-lock used in the nitrogen-15 R1 rho experiment.In the case of nitrogen-15, carbon-13-labeled samples include the line define LABEL_CN in the pulse program. Set P4 to the calculated pulse length. Use calcpowlev to calculate the power difference in decibels between P4 and the carbon-13 hard pulse.Copy the power level of the carbon-13 hard pulse to PLW4 and add the calculated power difference in decibels. Copy PLW4 to PLW2. To determine the appropriate relaxation delays for sampling run the first eight free induction decays, or FIDs, and process them.Choose vplist entries where the peak intensity of the longest delay experiment decreases to at least one over e, but not lower than 25%compared to the shortest delay experiment. Type rga in the command line to determine the receiver gain. Start a test run of the nitrogen-15 R1 rho experiment by typing zg in the command line.Check that the water signal is suppressed for all delays. Also, check increment nine, the second increment of the quadrature detection scheme.

331 Views.  Forschungszentrum Jülich.  Nuclear magnetic resonance (NMR) spectroscopy can characterize structural protein dynamics in a residue-specific manner. We provide a hands-on protocol for recording NMR 15N R1 and R2 relaxation and {1H}-15N heteronuclear Overhauser effect (hetNOE) experiments, sensitive to the picoseconds to nanoseconds timescale.

Protocol for NMR 15N Relaxation and hetNOE Experiments in Protein Dynamics Study

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