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<...

<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. 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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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2.5K ビュー.  Iowa State University. 高圧は、タンパク質立体構造サンプルの相対集団を変化させる方法です。高エネルギーの立体構造状態を特定し、特徴付けるための非常に有用です。pHや温度などのすべての摂動方法と比較すると、圧力摂動は適用しやすく、完全に可逆的です。また、主にガラスの空洞や空隙体積を有する領域に影響を与える局所的な摂動である。まず、ガラスピペットを使用し?...

ビデオ: 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...

nmr 15n relaxation experiments for the investigation of picosecond to nanoseconds structural dynamics of proteins

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...

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

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

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Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead...

15n cpmg relaxation dispersion for the investigation of protein conformational dynamics on the &#181;s-ms timescale

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the  &amp;#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...

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

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Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable...

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Building Langmuir Probes and Emissive Probes for Plasma Potential Measurements in Low Pressure, Low Temperature Plasmas

The main goal of this work is to make it easier for research groups unfamiliar with Langmuir probes and emissive probes to use them as plasma diagnostics, especially near plasma boundaries. We do this by demonstrating how to build the probes from readily available materials and...

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Running the NMR Relaxation Experiments on the Spectrometer

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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...

disentangling glycan-protein interactions: nuclear magnetic resonance (nmr) to the rescue

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.

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

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Low-Dose Computed Tomography (LDCT) Data Collection for Detecting Pulmonary Nodules  

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High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

The molecular structures and dynamics of solids, liquids, gases, and mixtures are of critical interest to diverse scientific fields. High-temperature, high-pressure in situ MAS NMR enables detection of the chemical environment of constituents in mixed phase systems under tightly controlled chemical...

administering and detecting protein marks on arthropods for dispersal research

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

monitoring protein-ligand interactions in human cells by real-time quantitative in-cell nmr using a high cell density bioreactor

Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor

This protocol describes the setup of an NMR bioreactor to keep encapsulated human cells viable for up to 72 h, followed by time-resolved in-cell NMR data acquisition and analysis. The methodology is applied to monitor intracellular protein-ligand interactions in real...

characterizing single-molecule conformational changes under shear flow with fluorescence microscopy

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow...

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Hyperpolarized Xenon for NMR and MRI Applications

The production of hyperpolarized xenon by means of spin exchange optical pumping (SEOP) is described. This method yields a ~10000-fold enhancement of the nuclear spin polarization of Xe-129 and has applications in nuclear magnetic resonance spectroscopy and imaging. Examples of gas phase and solution state experiments are...

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Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

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This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data...

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High Pressure Single Crystal Diffraction at PX^2

In this report, we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell at the GSECARS 13-BM-C beamline at the Advanced Photon Source. ATREX and RSV programs are used to analyze the...

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

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

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

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

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.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

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 ...

gP2S, an Information Management System for CryoEM Experiments

Cryogenic electron microscopy (cryoEM) has become an integral part of many drug-discovery projects because crystallography of the protein target is not always achievable and cryoEM provides an alternative means to support structure-based ligand design. When dealing with a large number of distinct projects, and within each project a potentially large number of ligand-protein co-structures, accurate record keeping rapidly becomes challenging. Many experimental parameters are tuned for each target, including at the sample preparation, grid preparation, and microscopy stages. Therefore, accurate record keeping can be crucially important to enable long-term reproducibility, and to facilitate efficient teamwork, especially when steps of the cryoEM workflow are performed by different operators. To help deal with this challenge, we developed a web-based information management system for cryoEM, called gP2S. 
The application keeps track of each experiment, from sample to final atomic model, in the context of projects, a list of which is maintained in the application, or externally in a separate system. User-defined controlled vocabularies of consumables, equipment, protocols and software help describe each step of the cryoEM workflow in a structured manner. gP2S is widely configurable and, depending on the team's needs, may exist as a standalone product or be a part of a broader ecosystem of scientific applications, integrating via REST APIs with project management tools, applications tracking the production of proteins or of small molecules ligands, or applications automating data collection and storage. Users can register details of each grid and microscopy session including key experimental metadata and parameter values, and the lineage of each experimental artifact (sample, grid, microscopy session, map, etc.) is recorded. gP2S serves as a cryoEM experimental workflow organizer that enables accurate record keeping for teams, and is available under an open-source license.

gP2S is a web application for the tracking of cryoEM experiments. Its main features are described, as are the steps required to install and configure the application. Once configured, the application allows one to accurately record metadata associated with negative stain and cryoEM experiments.

Cryogenic electron microscopy (cryoEM) has become an integral part of many drug-discovery projects because crystallography of the protein target is not ...

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.

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 ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represent the dynamic &#34;fingerprint&#34; of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known &#34;nanoruler&#34; to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations and mobility constants become accessible in cellular settings.
Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerability of the sample. Here we show how to perform this experiment, including the calibration steps using dual-color labeled &#946;2-adrenergic receptor (&#946;2AR) labeled with eGFP and SNAP-tag-TAMRA. A step-by-step data analysis procedure is provided using open-source software and templates that are easy to customize.
Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis at near-physiological conditions.

We present an experimental protocol and data analysis workflow to perform live cell dual-color fluorescence cross correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques.

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...