Name: high-pressure nmr experiments for detecting protein low-lying conformational states
Uploaded: 2021-06-29T14:30:00
Description: Watch this Scientific Journal Video about High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States at JoVE.com

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.
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	<li>Choice of buffer: Use equal mixture of anionic and cationic buffers, such as phosphate and Tris20<...

<ol>
	<li>Alderson, R. T., Kay, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unveiling+invisible+protein+states+with+NMR+spectroscopy.">Unveiling invisible protein states with NMR spectroscopy.</a> Current Opinion in Structural Biology. 60, 39-49 (2020).</li><li>Korzhnev, D. M., Kay, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=probing+invisible,+low-populated+states+of+protein+molecules+by+relaxation+dispersion+NMR+spectroscopy:+An+application+to+protein+folding.">probing invisible, low-populated states of protein molecules by relaxation dispersion NMR spectroscopy: An application to protein folding.</a> Accounts of Chemical Research. 41, 442-451 (2008).</li><li>Loria, P. J., Berlow, R. B., Watt, E. 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. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+exchange+saturation+transfer+(CEST):+an+efficient+tool+for+detecting+molecular+information+on+proteins'+behaviour.">Chemical exchange saturation transfer (CEST): an efficient tool for detecting molecular information on proteins' behaviour.</a> Analyst. 39, 2687-2690 (2014).</li><li>Fawzi, N. L., Ying, J., Torchia, D. A., Clore, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+exchange+kinetics+and+atomic+resolution+dynamics+in+high-molecular-weight+complexes+using+dark-state+exchange+saturation+transfer+NMR+spectroscopy.">Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy.</a> Nature Protocols. 7, 1523-1533 (2012).</li><li>Anthis, N. J., Clore, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+transient+dark+states+by+NMR+spectroscopy.">Visualizing transient dark states by NMR spectroscopy.</a> Quarterly Reviews of Biophysics. 48, 35-116 (2015).</li><li>Roche, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cavities+determine+the+pressure+unfolding+of+proteins.">Cavities determine the pressure unfolding of proteins.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 6945-6950 (2012).</li><li>Chen, C. R., Makhatadze, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+determinant+of+the+effects+of+hydrostatic+pressure+on+protein+folding+stability.">Molecular determinant of the effects of hydrostatic pressure on protein folding stability.</a> Nature Communications. 8, 14561 (2017).</li><li>Roche, J., Royer, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+pressure+denaturation+of+proteins.">Lessons from pressure denaturation of proteins.</a> Journal of the Royal Society Interface. 15, 20180244 (2018).</li><li>Xu, X., Gagn&#233;, D., Aramini, J. M., Gardner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+and+compressibility+differences+between+protein+conformations+revealed+by+high-pressure+NMR.">Volume and compressibility differences between protein conformations revealed by high-pressure NMR.</a> Biophysical Journal. 120, 924-935 (2021).</li><li>Akasaka, K., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-lying+excited+states+of+proteins+revealed+from+non-linear+pressure+shifts+in+1H+and+15N+NMR.">Low-lying excited states of proteins revealed from non-linear pressure shifts in 1H and 15N NMR.</a> Biochemistry. 40, 8665-8671 (2001).</li><li>Akasaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+conformational+fluctuation+of+proteins+by+pressure+perturbation.">Probing conformational fluctuation of proteins by pressure perturbation.</a> Chemical Reviews. 106, 1814-1835 (2006).</li><li>Kitahara, R., Yokoyama, S., Akasaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+snapshots+of+a+fluctuating+protein+structure:+ubiquitin+at+30+bar-3+kbar.">NMR snapshots of a fluctuating protein structure: ubiquitin at 30 bar-3 kbar.</a> Journal of Molecular Biology. 347, 277-285 (2005).</li><li>Roche, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=remodeling+of+the+folding+free+energy+landscape+of+Staphylococcal+nuclease+by+cavity-creating+mutations.">remodeling of the folding free energy landscape of Staphylococcal nuclease by cavity-creating mutations.</a> Biochemistry. 51, 9535-9546 (2012).</li><li>Nucci, N. V., Fuglestad, B., Athanasoula, E. A., Wand, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cavities+and+hydration+in+the+pressure+unfolding+of+T4+lysozyme.">Role of cavities and hydration in the pressure unfolding of T4 lysozyme.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 13846-13851 (2014).</li><li>Maeno, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cavity+as+a+source+of+conformational+fluctuation+and+high-energy+state:+High-pressure+NMR+study+of+a+cavity-enlarged+mutant+of+T4+lysozyme.">Cavity as a source of conformational fluctuation and high-energy state: High-pressure NMR study of a cavity-enlarged mutant of T4 lysozyme.</a> Biophysical Journal. 108, 133-145 (2015).</li><li>Peterson, R. W., Wand, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-contained+high-pressure+cell,+apparatus,+and+procedure+for+the+preparation+of+encapsulated+proteins+dissolved+in+low+viscosity+fluids+for+nuclear+magnetic+resonance+spectroscopy.">Self-contained high-pressure cell, apparatus, and procedure for the preparation of encapsulated proteins dissolved in low viscosity fluids for nuclear magnetic resonance spectroscopy.</a> Review of Scientific Instruments. 76, 094101 (2005).</li><li>Goddard, T. D., Kneller, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Sparky 3. , (2010).</li><li>Caro, J. A., Wand, J. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+volume+changes+in+pressure-induced+protein+unfolding.">Revisiting volume changes in pressure-induced protein unfolding.</a> Biochimica et Biophysica Acta. 1595, 201-209 (2002).</li><li>Erlach, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+nonliner+pressure-induced+chemical+shift+changes+and+thermodynamic+parameters.">Relationship between nonliner pressure-induced chemical shift changes and thermodynamic parameters.</a> Journal of Physical Chemistry B. 118, 5681-5690 (2014).</li><li>de Oliveira, G. A. P., Silva, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hypothesis+to+reconcile+the+physical+and+chemical+unfolding+of+proteins.">A hypothesis to reconcile the physical and chemical unfolding of proteins.</a> Proceedings of the National Academy of Sciences of The United States of America. 112, 2775-2784 (2015).</li><li>Nguyen, L. M., Roche, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+NMR+techniques+for+the+study+of+protein+dynamics,+folding+and+aggregation.">High-pressure NMR techniques for the study of protein dynamics, folding and aggregation.</a> Journal of Magnetic Resonance. 277, 179-185 (2017).</li></ol>

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 i...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 valu...

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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Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

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Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

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This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

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Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

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Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.

The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography.

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...