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<...

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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 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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2.5K Views.  Iowa State University. 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.