This work was supported by funds from the Roy J. Carver Charitable Trust to Julien Roche. We thank J. D. Levengood and B. S. Tolbert for kindly providing the RRM2 sample.

NOTE: The protocol described here requires (i) a high-pressure pump and cell with a 2.5 kbar rated aluminum-toughened zirconia tube18, (ii) the software SPARKY19 for analysis of the NMR spectra, and (iii) a curve fitting software.
1. Sample preparation, assembly of the high-pressure cell, and setting up the experiments.
<ol>
	<li>Choice of buffer: Use equal mixture of anionic and cationic buffers, such as phosphate and Tris20,21. 
	NOTE: The pKa of anionic buffers such as phosphate and MES is associated with a substantial reaction volume (i.e., the difference in the partial molal volumes of the acid and the ionized products). The pH of such buffers can therefore be significantly affected by a change of pressure (~0.25-0.5 pH unit/kbar).</li>
	<li>Ensure that the required sample volume is similar to that of a standard 3 mm diameter NMR tube (~300&#956;L).</li>
	<li>Introduce the 15N-labeled sample with a glass pipette into the zirconia tube. Make sure the sample seats at the bottom of the tube. Complete with 200 &#956;L of mineral oil to prevent the sample from mixing with the transmission liquid (e.g., water). Fill the rest of the tube with transmission liquid.</li>
	<li>Put a single-use O-ring on top of the zirconia tube and slide the tube into the base (Figure 1A,B). Then, connect the tube to the high-pressure tether line and tighten the base to the cell first by hand. Then, apply 14.7 Nm of torque to prevent leaks at lower pressure (Figure 1C,D).</li>
	<li>To check the integrity of the pressure cell assembly, pressurize the tube up to 300 bar outside the spectrometer using cell support and containment vessel. Wait for 15 min, reset the pressure to 1 bar and check for leaks with a clean lint-free wipe.</li>
	<li>Insert the unpressurized tube into the spectrometer by carefully guiding the tether line. Slide the tube in the spectrometer until reaching the sample sitting position (Figure 1E).</li>
	<li>Lock, shim, match, and tune the 1H and 15N channels as usual. 
	NOTE: Shims for high-pressure rated zirconia tubes are very different from standard NMR tubes. It is recommended to save the optimized shims for future use.</li>
	<li>Set up a 1H-15N-HSQC or TROSY-HSQC and record a reference experiment at atmospheric conditions (1bar).</li>
</ol>
2. Recording high-pressure NMR experiments
<ol>
	<li>Gradually increase the pressure from 1 bar to 2.5 kbar with 500 bar increments to test the overall stability of the protein. Set the speed of the pressure pump by default at ~18 bar/s. If the precise folding/unfolding rates are not known, let the sample equilibrate 15-20 min after each 500-bar increment. Record a spectrum at 2.5 kbar.</li>
	<li>Gradually decrease the pressure back to 1 bar with 500 bar steps to test the reversibility of the pressure perturbation. Record another spectrum at atmospheric conditions and compare the chemical shifts and peak intensities with that of the reference spectrum previously recorded in the same conditions. 
	NOTE: If the native crosspeaks are more intense after the pressure run, it may indicate that small aggregates present in solution at atmospheric pressure may have dissociated and properly refolded. On the other hand, a loss in the intensity or significant chemical shift changes suggest that the protein may experience a non-reversible misfolding in high-pressure conditions.</li>
	<li>Record a series of 2D experiments from 1 bar to 2.5 kbar every 500 bar. It is recommended to record additional experiments near the inflection point of the folding/unfolding transition to improve the precision of the fit.</li>
</ol>
3. Analyzing peak intensity changes
<ol>
	<li>Process all the spectra and transfer the backbone assignment from the reference spectrum at 1 bar to the spectrum recorded at 500 bar, and then transfer the assignment from 500 bar to 1 kbar and so forth. 
	NOTE: Because pressure induces a non-uniform shift of 1H and 15N chemical shifts, do not simply copy the backbone assignment from one spectrum to the next. Adjust it manually.</li>
	<li>In Sparky menu, click on Peak &#62; Peak List (lt). In the Peak List window, click on Options and select the option to display both Frequencies (ppm) and Data Height. Save the list obtained for each spectrum.</li>
	<li>In a curve fitting software, copy the crosspeak identity and peak intensity values to have the pressure values (in bar) as the X-axis variable and the intensity as the Y-axis variable.</li>
	<li>If a complete or near complete (&#62;80%) unfolding is observed, fit the individual peak intensity profiles to extract the free energy and volume change upon unfolding, respectively, using a simple two-state model: 
	<img alt="Equation 1" src="/files/ftp_upload/62701/62701eq01.jpg" />&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Eq. 1 
	Where, &#34;I&#34; is the observed intensity of a crosspeak at a given pressure p and IF is the intensity of the same crosspeak in a fully folded state. R is the gas constant, T is the absolute temperature, &#916;GU0 the standard Gibbs free energy difference between the unfolded and folded states at atmospheric pressure p0 (1 bar), and &#916;Vu the volume change upon unfolding. With the pressure p in bar and temperature T in kelvin, R = 1.987 cal/K, &#916;GU0 is in cal/mol, and &#916;VU is in cal/mol/bar. Multiply the &#916;VU values obtained from the fit by 41.84 to convert into mL/mol. &#916;VU values are typically in the range of -50 to -150 mL/mol for globular proteins22. Here, all the parameters are expressed with regard to the unfolding reaction but can easily be converted into folding reaction parameters (&#916;GU0 = -&#916;GF0 and &#916;VU = -&#916;VF).</li>
</ol>
4. Analyzing chemical shift changes
<ol>
	<li>Arrange the columns in the software in order to have pressure points as variable and the 1H chemical shifts extracted from Sparky lists as the Y-axis.</li>
	<li>Fit the pressure dependence of the 1H chemical shifts to a simple quadratic equation: 
	&#948;(p) = &#948;0(p0) + B1(p-p0) + B2 (p-p0)2&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;&#160;Eq. 2 
	Where, &#948;(p) is the measured 1H chemical shift of a crosspeak at a given pressure p and &#948;0(p0) the 1H chemical shifts of the same crosspeak in the reference spectrum recorded at 1 bar. B1 and B2 represent the first and second-order parameters expressed in ppm/bar and ppm/bar2, respectively.</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+enzyme+motions+by+solution+NMR+relaxation+dispersion.">Characterization of enzyme motions by solution NMR relaxation dispersion.</a> Accounts of Chemical Research. 41, 214-221 (2008).</li><li>Ishima, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CPMG+relaxation+dispersion.">CPMG relaxation dispersion.</a> Methods in Molecular Biology. 1084, 29-49 (2014).</li><li>Longo, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+aspects+of+high-pressure+NMR+spectroscopy+and+its+applications+in+protein+biophysics+and+structural+biology.">Practical aspects of high-pressure NMR spectroscopy and its applications in protein biophysics and structural biology.</a> Methods. 148, 67-80 (2018).</li><li>Kitamura, T., Itoh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+volume+of+protonic+ionization+for+buffering+agents.+Prediction+of+pressure+dependence+of+pH+and+pOH.">Reaction volume of protonic ionization for buffering agents. Prediction of pressure dependence of pH and pOH.</a> Journal of Solution Chemistry. 16, 715-725 (1987).</li><li>Royer, C. 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All the authors have read and approved the manuscript. They declare no conflicts of interest.

This&#160;study details a protocol implemented to probe protein structural and thermodynamics responses to pressure perturbation. The high-pressure experiments recorded here on RRM2 demonstrate that large variations in &#916;VU values, indicative of non-fully cooperative unfolding, can be found in a relatively small single domain protein. A similar picture emerges from the analysis of 1H chemical shift changes under pressure. It should be noted Kalbitzer and coworkers have demonstrated that a more in-depth analysis of chemical shift changes can be performed linking the ratio between the non-linear and linear coefficients (B2/B1) with the ratio of the change in compressibility and volume (&#916;&#946;/&#916;V)23. These results highlight the ability of high hydrostatic pressure to destabilize individual structural motifs and subdomains based on the local thermodynamic stability and volume change associated with unfolding. Recording a simple set of pressure titration experiments can therefore provide valuable information regarding the folding mechanisms and subdomain architecture of a protein.
The whole pressure system (pump and controller) is relatively compact and can be installed on a standard utility cart for easy access to the spectrometer (Figure 1). All pulse sequences can be used under pressure without modification, including triple resonance experiments. The only limitation is that pressure-rated zirconia tubes reduce the sensitivity of NMR experiments by almost 50% compared to a standard 3 mm diameter NMR tube. This may present a challenge for protein samples that can&#39;t be concentrated to a high-enough level. Compared to other perturbation methods such as temperature or chemical denaturants, hydrostatic pressure presents the advantage of being, with very few exceptions, fully reversible. Because pressure favors the dissociation of protein complexes, there is, for example, very little risk of sample aggregation, a problem often encountered with high temperatures.
While hydrostatic pressure is a powerful tool for studying protein stability, it is important to keep in mind that there is no perfect method of perturbation. Each of them highlights the specificity of a protein free-energy landscape in a different way. For example, if two conformational states have the same molar volume, an increase in hydrostatic pressure will not significantly change their relative populations. Similarly, if two conformational states have a similar exposed surface area, adding chemical denaturants would not necessarily increase the relative population of one state compared to the other one. Ideally, different perturbation methods should be combined to obtain a complete description of a protein free-energy landscape15,24. Finally, it should be noted that although the manuscript is focused on the pressure dependence of intensity and chemical shifts from series of 2D 1H-15N experiments measured at equilibrium, a wide range of NMR experiments can be coupled to pressure perturbation to probe different aspects of the stability, the folding mechanism, and the conformational dynamics of proteins and proteins complexes20,25.

It has long been recognized that higher-energy, sparsely populated conformational states of proteins and protein complexes play a key role in many biological pathways1,2,3. Thanks to experiments based on Carr-Purcell-Meiboom-Gill (CPMG)4, Chemical Exchange Saturation Transfer (CEST)5, and dark-state exchange saturation transfer (DEST)6 pulse sequences (among others), solution NMR spectroscopy has emerged as a method of choice for characterizing transient conformational states7. Along with these experiments, perturbations such as temperature, pH, or chemical denaturants can be introduced to increase the relative population of higher energy conformational substates. Similarly, protein equilibria can also be perturbed by applying high hydrostatic pressure. Depending on the magnitude of the volume change associated with the corresponding conformational changes, an increase of pressure by a few hundred to a few thousand bars can significantly stabilize a higher energy state or cause a protein to completely unfold8,9,10. Protein NMR spectra typically display two types of changes with hydrostatic pressure: (i) chemical shift changes and (ii) peak intensity changes. Chemical shift changes reflect changes at the protein surface-water interface and/or local compression of the protein structure on a fast time scale (relative to NMR time scale)11. Crosspeaks exhibiting large non-linear chemical shifts pressure dependence can indicate the presence of higher energy conformational states12,13. On the other hand, peak intensity changes point to major conformational transitions on a slow time scale, such as changes in folded/unfolded state populations. The presence of folding intermediates or higher energy states can be detected from large variations in the magnitude of the volume change upon unfolding measured for different residues of a given protein14,15,16,17. Based on our experience, even small proteins that are typically classified as two-state folders exhibit non-uniform responses to pressure, which provides useful information about their local folding stability. Described here is a protocol for the acquisition and analysis of amide peak intensity and 1H chemical shifts pressure dependence, using as a model protein the isolated RNA recognition motif 2 (RRM2) of the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1).

<table><tbody><tr><td>Bruker Nmr Cell 2.5 Kbar</td><td>Daedalus Innovations LLC</td><td>NMRCELL-B</td><td></td></tr><tr><td>Sparky3</td><td>University of California San Francisco, CA</td><td>N/A</td><td></td></tr><tr><td>Xtreme-60 Syringe pump</td><td>Daedalus Innovations LLC</td><td>XTREME-60</td><td></td></tr></tbody></table>

high-pressure nmr experiments for detecting protein low-lying conformational states

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible manner. Hydrostatic pressure drives thermodynamical equilibria toward the state(s) with the lower molar volume. Increasing pressure offers, therefore, the opportunities to finely tune the stability of globular proteins and the oligomerization equilibria of protein complexes. High-pressure NMR experiments allow a detailed characterization of the factors governing the stability of globular proteins, their folding mechanisms, and oligomerization mechanisms by combining the fine stability tuning ability of pressure perturbation and the site resolution offered by solution NMR spectroscopy. Here we present a protocol to probe the local folding stability of a protein via a set of 2D 1H-15N experiments recorded from 1 bar to 2.5 kbar. The steps required for the acquisition and analysis of such experiments are illustrated with data acquired on the RRM2 domain of hnRNPA1.

The protocol described here was used to probe the pressure dependence of RRM2, the second RNA recognition motif of hnRNPA1 (residues 95-106), which is almost completely unfolded within the 2.5 kbar range (&#62;90%). 1H-15N spectra were collected at 1 bar, 500 bar, 750 bar, 1 kbar, 1.5 kbar, 2 kbar, and 2.5 kbar&#160;(Figure 2). Since none of the native crosspeaks were visible above the noise level at 2.5 kbar, all corresponding residues were attributed an intensity value of 0 at this pressure (Figure 3A). A total of 45 individual pressure intensity profiles were fitted according to Equation 1 (Eq. 1) to obtain the corresponding changes in standard Gibbs free-energy (&#916;GU0) and volume (&#916;VU) associated with the unfolding reaction (Figure 3A). The calculated &#916;VU values ranged from -41 to -88 mL/mol and &#916;GU0 from 1.8-3.3 kcal/mol. When mapped on the domain structure (pdb 1U1R), it appears that residues with the largest magnitude of volume change are found within the domain structural core (in red, in Figure 3B), while those with the smallest magnitude of volume change are mainly located in the loop connecting the &#946;-strands 2 and 3, and the loop connecting &#946;-strand 4 to the C-terminal helix (in green, Figure 3B). The pressure dependence of the native backbone 1H chemical shifts was also analyzed to probe the degree of compressibility and conformational heterogeneity of the folded state ensemble. Individual profiles of 1H chemical shifts as a function of pressure were fitted using Eq. 2 in order to extract site-specific linear (B1) and non-linear (B2) coefficients (Figure 3C). The residues with the largest non-linear coefficients were mostly found in the structural core of the domain (in yellow, Figure 3D), while residues with the smallest non-linear coefficients were mostly located within loops connecting the different structural motifs (in blue, Figure 3D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62701/62701fig01.jpg" /> 
Figure 1: High-pressure cell assembly and installation in the spectrometer. (A-D) The full high-pressure cell assembly requires a zirconia tube, a single-use O-ring, the cell base (A,B). The tube is connected to the tether line by tightening the base to the cell (C,D). 14.7 Nm of torque is required to prevent leaks at lower pressure. (E) The zirconia tube connected to the Xtreme-60 Syringe pump via the tether line is introduced into the spectrometer until reaching the normal sample sitting position. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62701/62701fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62701/62701fig02.jpg" /> 
Figure 2: Unfolding of hnRNPA1 RRM2 domain under pressure. 1H-15N HSQC spectra collected at (A) 1 bar, (B) 1,000 bar, (C) 1,500 bar, (D) and 2,500 bar show a complete unfolding of the RRM2 domain under pressure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62701/62701fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62701/62701fig03.jpg" /> 
Figure 3: Analysis of RRM2 peak intensity and 1H chemical shifts pressure dependence. (A) Representative peak intensity profiles recorded for RRM2 between 1 and 2,000 bar. Solid lines represent the fit obtained using Eq. 1 to calculate the corresponding standard free-energy and volume changes associated with the unfolding reaction. (B) The top 25% of residues with the largest measured magnitude of &#916;VU are highlighted with red spheres on RRM2 reference structure (pdb 1U1R). Residues with the smallest magnitude of &#916;VU are shown in green. (C) Representative changes of 1H chemical shifts measured for RRM2 between 1 bar and 2,000 bar. Solid lines represent the fit to Eq. 2 to calculate the linear (B1) and non-linear (B2) pressure coefficients. (D) The top 25% of residues with the largest B2 coefficients are shown in yellow, while those with the smallest B2 coefficients are shown in blue. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62701/62701fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States at JoVE.com

High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States

We provide a detailed description of the steps required to assemble a high-pressure cell, set up and record high-pressure NMR experiments, and finally analyze both peak intensity and chemical shift changes under pressure. These experiments can provide valuable insights into the folding pathways and structural stability of proteins.

High-pressure is a well-known perturbation method that can be used to destabilize globular proteins and dissociate protein complexes in a reversible ...

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2.6K Views.  Iowa State University. Hoge druk is de methode bij uitstek om de relatieve populatie van een eiwitconformatiemonster te veranderen. Het is uiterst nuttig voor het isoleren en karakteriseren van hoogenergetische conformatietoestanden. Vergeleken met alle perturbatiemethoden, zoals pH of temperatuur, is drukperturbatie gemakkelijk toe te passen en volledig omkeerbaar.Het is ook een lokale verstoring, die voornamelijk regio's met glazen holtes of lege volumes treft. Om te beginnen introduceert u het stikstof-15...

Video: High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

High-Sensitivity Nuclear Magnetic Resonance at Giga-Pascal Pressures: A New Tool for Probing Electronic and Chemical Properties of Condensed Matter under Extreme Conditions

Nuclear Magnetic Resonance (NMR) is one of the most important techniques for the study of condensed matter systems, their chemical structure, and their electronic properties. The application of high pressure enables one to synthesize new materials, but the response of known materials to high pressure is a very useful tool for studying their electronic structure and developing theories. For example, high-pressure synthesis might be at the origin of life; and understanding the behavior of small molecules under extreme pressure will tell us more about fundamental processes in our universe. It is no wonder that there has always been great interest in having NMR available at high pressures. Unfortunately, the desired pressures are often well into the Giga-Pascal (GPa) range and require special anvil cell devices where only very small, secluded volumes are available. This has restricted the use of NMR almost entirely in the past, and only recently, a new approach to high-sensitivity GPa NMR, which has a resonating micro-coil inside the sample chamber, was put forward. This approach enables us to achieve high sensitivity with experiments that bring the power of NMR to Giga-Pascal pressure condensed matter research. First applications, the detection of a topological electronic transition in ordinary aluminum metal and the closing of the pseudo-gap in high-temperature superconductivity, show the power of such an approach. Meanwhile, the range of achievable pressures was increased tremendously with a new generation of anvil cells (up to 10.1 GPa), that fit standard-bore NMR magnets. This approach might become a new, important tool for the investigation of many condensed matter systems, in chemistry, geochemistry, and in physics, since we can now watch structural changes with the eyes of a very versatile probe.

Nuclear magnetic resonance is one of the most important spectroscopic tools. Here, the development of a new approach under high pressure, currently up to 10.1 GPa, is presented. This opens a new window into condensed matter physics and chemistry, where high-pressure research is of great importance.

Nuclear Magnetic Resonance (NMR) is one of the most important techniques for the study of condensed matter systems, their chemical structure, and their ...

Engineering

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the &#181;s-ms Timescale

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 &#181;s-ms timescale. Here, the study presents detailed protocols for acquisition and analysis of a&#160;15N relaxation dispersion experiment. As an example, the pipeline for the analysis of the &#181;s-ms dynamics in the C-terminal domain of bacteria Enzyme I is shown.

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the  &#181;s-ms Timescale

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 ...

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

Chemistry

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

Advanced NMR Techniques for Glycan-Protein Interaction Analysis

Disentangling Glycan-Protein Interactions: Nuclear Magnetic Resonance (NMR) to the Rescue

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and their biological consequences are intimately related to the three-dimensional structures of both partners, as well as to their dynamic features and their presentation on the corresponding cell compartments. NMR techniques are unique to disentangle these characteristics and, indeed, diverse NMR-based methodologies have been developed and applied to monitor the binding events of glycans with their associate receptors. This protocol outlines the procedures to acquire, process and analyze two of the most powerful NMR methodologies employed in the NMR-glycobiology field, 1H-Saturation transfer difference (STD) and 1H,15N-Heteronuclear single quantum coherence (HSQC) titration experiments, which complementarily offer information from the glycan and protein perspective, respectively. Indeed, when combined they offer a powerful toolkit for elucidating both the structural and dynamic aspects of molecular recognition processes. This comprehensive approach enhances our understanding of glycan-protein interactions and contributes to advancing research in the chemical glycobiology field.

Author Spotlight: Unveiling the Structural and Dynamic Aspects of Glycan Molecular Recognition

Here, we present a protocol detailing the acquisition, processing, and analysis of a series of NMR experiments aimed at characterizing protein-glycan interactions in solution. Most common ligand-based and protein-based methodologies are outlined, which undoubtedly contribute to the fields of structural glycobiology and molecular recognition studies.

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and ...

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

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

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 ...

Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions

Source: Winkelaar, G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58537">Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST).</a> J. Vis. Exp. (2018).
This video describes the nuclear magnetic resonance spectroscopy technique to study protein-protein interactions between 15N-labeled wild-type and mutant envoplakin proteins and the unlabeled vimentin protein. The successful interaction between wild-type envoplakin and vimentin leads to extensive line broadening and peak disappearance in the NMR spectra, whereas the absence of an interaction between the mutated envoplakin and vimentin results in well-resolved peaks in the NMR spectra.

Source: Winkelaar, G., et al. Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale ...

High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States

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