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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</table>

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

Research

JoVE Journal

Biochemistry

337 vistas.  Forschungszentrum Jülich. Investigamos la dinámica estructural de las proteínas mediante espectroscopía de RMN para comprender mejor la función molecular. RMN son las siglas de Espectroscopia de Resonancia Magnética Nuclear. Reduce el efecto de que los espines nucleares de los átomos de hidrógeno pueden hacer la transición entre dos estados energéticamente diferentes y un campo magnético automático.Podemos utilizar la espectroscopia de RMN para estudiar la dinámica ...