Name: defining hsp33's redox-regulated chaperone activity and mapping conformational changes on hsp33 using hydrogen-deuterium exchange mass spectrometry
Uploaded: 2018-06-07T15:00:00
Description: Watch this Scientific Journal Video about Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry at JoVE.co

The authors are thankful to Meytal Radzinski for her helpful discussions and critical reading of the article, and to Patrick Griffin and his lab members for their unlimited assistance while establishing the HDX analysis platform. The authors are grateful to the German-Israel Foundation (I-2332-1149.9/2012), the Binational Science Foundation (2015056), the Marie-Curie integration grant (618806), the Israel Science Foundation (1765/13 and 2629/16), and the Human Frontier Science Program (CDA00064/2014) for their financial support.

1. Preparation of Fully Reduced and Fully Oxidized Proteins
<ol>
	<li>Preparation of a fully reduced protein 
	Note: Here, we describe the reduction of a zinc-containing protein, and use a ZnCl2 solution to restore the Zn-incorporated, reduced protein state. The ZnCl2 solution can be replaced or discarded. Note that the time and temperature of the reduction process depends on the protein stability and function, and is thus specific per protein.
	<ol>
		<li>Thaw the protein sa...

In this paper, we provided protocols for the analysis of redox-dependent chaperone activity and the characterization of structural changes upon the binding of a client protein. These are complementary methodologies to define potential chaperone-substrate complexes and analyze potential interaction sites.
Here, we applied these protocols for the characterization of a complex between the redox-regulated chaperone Hsp33 with a well-studied chaperone substrate CS. We presented two different types ...

Cells frequently encounter an accumulation of reactive oxygen species (ROS) produced as byproducts of respiration1,2, protein and lipid oxidation3,4, and additional processes5,6,7. Despite ROS&#39; beneficial role in diverse biological processes such as cellular signaling8,9 and immune response10, an imbalance between ROS production and its detoxification might occur, leading to oxidative stress7. The biological targets of ROS are proteins, lipids, and nucleic acids, the oxidation of which affect their structure and function. Therefore, the accumulation of cellular oxidants is strongly linked to a diverse range of pathologies including cancer9,11, inflammation12,13, and aging14,15, and have been found to be involved in the onset and progression of neurodegenerative disorders such as Alzheimer&#39;s, Parkinson&#39;s, and ALS disease16,17,18.
Both newly synthesized and mature proteins are highly sensitive to oxidation due to the potentially harmful modifications of their side chains, which shape protein structure and function19,20. Therefore, oxidative stress usually leads to a widespread protein inactivation, misfolding and aggregation, eventually leading to cell death. One of the elegant cellular strategies to cope with the potential damage of protein oxidation is to utilize redox-dependent chaperones, which inhibit the widespread protein aggregation, instead of forming stable complexes with misfolded client proteins21,22,23. These first-line defense chaperones are rapidly activated by a site-specific oxidation (usually on cysteine residues) that converts them into potent anti-aggregation molecules24. Since oxidative stress results in the inhibition of respiration and in decreases in the cellular ATP levels25, canonical ATP-dependent chaperones are less effective during oxidative stress conditions25,26,27. Therefore, redox-activated ATP-independent chaperones play a vital role in maintaining protein homeostasis upon the accumulation of oxidants in bacteria and eukaryotes (e.g., Hsp3328 and RidA29 in bacteria, Get330 in yeast, peroxiredoxins31 in eukaryotes). The activity of these chaperones strongly depends on reversible structural conformational changes induced by a site-specific oxidation that uncovers hydrophobic regions involved in the recognition of misfolded client proteins.
Research of the anti-aggregation mechanism and the principles governing the recognition of the client proteins by chaperones is not easy due to the dynamic and heterogenic nature of chaperone-substrate interactions32,33,34,35,36,37. However, stress-regulated chaperones have an opportunity to advance our understanding of the anti-aggregation function due to their ability to: 1) obtain two different forms of the chaperone, active (e.g., oxidized) and inactive (e.g., reduced), with the introduction or removal of a stress condition easily switching between them (e.g., oxidant and reducing agent), 2) have a broad range of substrates, 3) form highly stable complexes with the client proteins that may be evaluated by different structural methodologies, and 4) focus solely on the substrate recognition and release, mediated by redox-dependent conformational changes, as the majority of these chaperones lacks the folding capability.
Here, we analyze the bacterial redox-regulated chaperone Hsp33&#39;s anti-aggregation activity, a vital component of the bacterial defense system against oxidation-induced protein aggregation28. When reduced, Hsp33 is a tightly folded zinc-binding protein with no chaperone activity; however, when exposed to oxidative stress, Hsp33 undergoes extensive conformational changes which expose its substrate binding regions38,39. Upon oxidation, the zinc ion that is strongly bound to four highly conserved cysteine residues of the C-terminal domain is released40. This results in the formation of two disulfide bonds, an unfolding of the C-terminal domain, and a destabilization of the adjacent linker region41. The C-terminal and linker regions are highly flexible and are defined as intrinsically or partially disordered. Upon return to non-stress conditions, the cysteines become reduced and the chaperone returns to its native folded state with no anti-aggregation activity. The refolding of the chaperone leads to a further unfolding and destabilization of the bound client protein, which triggers its transfer to the canonical chaperone system, DnaK/J, for refolding38. Analysis of Hsp33&#39;s interaction sites suggests that Hsp33 uses both its charged disordered regions as well as the hydrophobic regions on the linker and N-terminal domain to capture misfolded client proteins and prevent their aggregation38,42. In the folded state, these regions are hidden by the folded linker and C-terminal domain. Interestingly, the linker region serves as a gatekeeper of Hsp33&#39;s folded and inactive state, &#34;sensing&#34; the folding status of its adjacent C-terminal domain34. Once destabilized by mutagenesis (either by a point mutation or a full sequence perturbation), Hsp33 is converted to a constitutively active chaperone regardless the redox state of its redox-sensitive cysteines43.
The protocols presented here allow monitoring of Hsp33&#39;s redox-dependent chaperone activity, as well as mapping conformational changes upon the activation and binding of client proteins. This methodology can be adapted to research other chaperone-client recognition models as well as non-chaperone protein-protein interactions. Moreover, we present protocols for the preparation of fully reduced and oxidized chaperones that can be used in studies of other redox-switch proteins, to reveal potential roles of protein oxidation on the protein activity.
Specifically, we describe a procedure to monitor chaperone activity in vitro and define its substrate specificity under different types of protein aggregation (chemically or thermally induced) using light scattering (LS) measured by a fluorospectrometer44. During aggregation, light scattering at 360 nm increases rapidly due to the increasing turbidity. Thus, aggregation can be monitored in a time-dependent manner at this wavelength. LS is a fast and sensitive method for testing protein aggregation and thus the anti-aggregation activity of a protein of interest using nanomolar concentrations, enabling the characterization of protein aggregation-related kinetic parameters under different conditions. Moreover, the LS protocol described here does not require expensive instrumentation, and can be easily established in any laboratory.
Nevertheless, it is quite challenging to obtain &#34;clean&#34; kinetic curves and to derive a protein&#39;s kinetic parameters from such light scattering experiments, due to noise and the large number of outliers generated by air bubbles and large aggregates. To overcome this obstacle, we present a novel graphical tool, Kfits45, used for reducing noise levels in different kinetic measurements, specifically fitted for protein aggregation kinetic data. This software provides preliminary kinetic parameters for an early assessment of the results and allows the user to &#34;clean&#34; large quantities of data quickly without affecting its kinetic properties. Kfits is implemented in Python and available in open source at 45.
One of the challenging questions in the field relates to mapping interaction sites between chaperones and their client proteins and understanding how chaperones recognize a wide range of misfolded substrates. This question is further complicated when studying highly dynamic protein complexes which involve intrinsically disordered chaperones and aggregation-prone substrates. Fortunately, structural mass spectrometry has dramatically advanced over the last decade and has successfully provided helpful approaches and tools to analyze the structural plasticity and map residues involved in protein recognition46,47,48,49. Here, we present one such technique-hydrogen-deuterium exchange mass spectrometry (HDX-MS)-which allows the mapping of residue-level changes in a structural conformation upon protein modification or protein/Ligand binding35,50,51,52,53,54,55. HDX-MS uses the continuous exchange of backbone hydrogens by deuterium, the rate of which is affected by the chemical environment, accessibility, and covalent and non-covalent bonds56. HDX-MS tracks these exchange processes using a deuterated solvent, commonly heavy water (D2O), and allows measurement based on the change in molecular weight following the hydrogen to deuterium exchange. Slower rates of hydrogen-deuterium exchange can result from hydrogens participating in hydrogen bonds or, simply, from steric hindrance, which indicates local changes in structure57. Changes upon a ligand binding or post-translational modifications can also lead to differences in the hydrogen environment, with a binding resulting in differences in the hydrogen-deuterium exchange (HDX) rates46,53.
We applied this technology to 1) map Hsp33 regions which rapidly unfold upon oxidation, leading to the activation of Hsp33, and 2) define the potential binding interface of Hsp33 with its full-length misfolded substrate, citrate synthase (CS)38.
The methods described in this manuscript can be applied to study redox-dependent functions of proteins in vitro, defining anti-aggregation activity and the role of structural changes (if any) in protein function. These methodologies can be easily adapted to diverse biological systems and applied in the laboratory.

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			<td height="21" style="height:21px;width:792px;">Acetonitrile HPLC plus</td>
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			<td>solvent</td>
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			<td height="20" style="height:20px;width:792px;">Tris(hydroxymethyl)aminomethane</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">252859</td>
			<td>buffer</td>
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			<td height="20" style="height:20px;width:792px;">Trifluoroacetic acid</td>
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			<td style="width:119px;">76-05-1</td>
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			<td style="width:256px;">Sigma Aldrich</td>
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			<td>solvent</td>
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			<td height="20" style="height:20px;">ZnCl2, Zinc Chloride</td>
			<td style="width:256px;">Merck</td>
			<td style="width:119px;">B0755416 308</td>
			<td>reagent</td>
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			<td height="20" style="height:20px;">DTT</td>
			<td>goldbio</td>
			<td>27565-41-9</td>
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			<td height="20" style="height:20px;">PD mini trap G-25 columns GE healthcare</td>
			<td>GE healthcare</td>
			<td>29-9180-07</td>
			<td>desalting column</td>
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			<td height="20" style="height:20px;">Potassium Phosphate</td>
			<td>United states Biochemical Corporation</td>
			<td>20274</td>
			<td>buffer</td>
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			<td height="40" style="height:40px;">Hydrogen peroxide 30%</td>
			<td style="width:256px;">Merck</td>
			<td style="width:119px;">K46809910526</td>
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			<td height="20" style="height:20px;">citrate synthase</td>
			<td style="width:256px;">sigma aldrich</td>
			<td style="width:119px;">C3260</td>
			<td>substrate</td>
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			<td style="width:256px;">sigma aldrich</td>
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			<td>denaturant</td>
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			<td height="20" style="height:20px;">Deuterium Chloride Solution</td>
			<td style="width:256px;">sigma aldrich</td>
			<td style="width:119px;">543047-10G</td>
			<td>buffer</td>
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			<td height="20" style="height:20px;">Deuterium Oxide 99%</td>
			<td style="width:256px;">sigma aldrich</td>
			<td style="width:119px;">151882-100G</td>
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			<td height="21" style="height:21px;width:792px;">Jasco FP-8500 Fluorospectrometer</td>
			<td>Jasco</td>
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			<td style="width:256px;">Sartorius</td>
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			<td height="23" style="height:23px;">AffiPro Immobilized Pepsin column (20mm length, 2.0mm diameter).</td>
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			<td height="20" style="height:20px;">Waters Pre-column (ACQUITY UPLC BEH C18 VanGuard 130 &#197;, 1.7um, 2.1mmx5mm)</td>
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			<td style="width:119px;"></td>
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			<td height="20" style="height:20px;width:792px;">Vinyl Anaerobic chamber with Airlock door</td>
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			<td height="22" style="height:22px;width:792px;">PAL system LHX - robotic system for handling HDX samples</td>
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			<td colspan="2">https://www.palsystem.com/index.php?id=840</td>
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			<td height="22" style="height:22px;width:792px;">Dionex Ultimate 3000, XRS pump</td>
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			<td height="22" style="height:22px;width:792px;">Dionex AXP-MS auxiliary pump</td>
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			<td height="21" style="height:21px;width:792px;">Kfits: Fit aggregation Data</td>
			<td>http://kfits.reichmannlab.com/fitter/</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Thermo Scientific Xcalibur software</td>
			<td style="width:256px;">https://www.thermofisher.com/order/catalog/product/OPTON-30487</td>
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			<td height="80" style="height:80px;">Q Exactive MS Series Tune Interface (Tune)</td>
			<td style="width:256px;">https://tools.thermofisher.com/content/sfs/brochures/WS-MS-Q-Exactive-Calibration-Maintenance-iQuan2016-EN.pdf</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="60" style="height:60px;">Chronos software (Axel Semrau)</td>
			<td style="width:256px;">http://www.axel-semrau.de/en/Software/Software+Solutions/Chronos-p-966.html</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="40" style="height:40px;">Proteome Discoverer V1.4 software</td>
			<td style="width:256px;">https://www.thermofisher.com/order/catalog/product/OPTON-30795</td>
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			<td></td>
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			<td height="24" style="height:24px;">HDX workbench software</td>
			<td colspan="3">http://hdx.florida.scripps.edu/hdx_workbench/Home.html</td>
		</tr>
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defining hsp33's redox-regulated chaperone activity and mapping conformational changes on hsp33 using hydrogen-deuterium exchange mass spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

The two methods presented make it possible to follow the kinetic activity and dynamics of protein interactions between a chaperone and its substrate. Moreover, the reduction-oxidation protocol allows the preparation of a fully reduced and fully oxidized chaperone, giving a more in-depth understanding of the activation mechanism of redox-dependent disordered chaperones.
First, we used light scattering in order to examine the redo...

Watch this Scientific Journal Video about Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry at JoVE.co

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

The workflow presented here can help extend our understanding of the cellular role of chaperones during oxidative stress conditions. Specifically, it can help to understand how do chaperones prevent protein aggregation during unfolding conditions. For these two backs of different techniques provide insight into the activity of flag segregated chaperones.It can also be used to study other protein for interaction such as enzyme social interactions and protein dynamics. Visual demonstration of this method is critical as it involves the usage of different instruments which may be challenging to new users. First, spin down a previously thawed protein sample to remove aggregates.Then add five millimolar DTT and 20 micromolar zinc chloride and incubate the sample for at least 1.5 hours at 37 degrees Celsius. To remove the DTT equilibrate a desalting column with at 40 millimolar potassium phosphate buffer by filling the column completely with the buffer and letting it drip out. Using tweezers, gently push down on the white disc filter in the column and remove it.Refill the column with potassium phosphate buffer and centrifuge at 1000 times G for three minutes. Following centrifugation transfer the column into a clean tube and add the protein sample slowly to the middle of the column. Centrifuge the column at 1000 times G for two minutes so that the DTT-free protein is in the flow-through.Now check the protein concentration and measure its absorbance at 280 nanometers. Distribute half of the protein samples into aliquots. Incubate the aliquots under anaerobic conditions for 20 minutes for complete removal of oxygen.Then seal the tubes with plastic film and store the samples under cold conditions. Next, add freshly-prepared five millimolar hydrogen peroxide to the remaining protein sample and incubate for three hours at 40 degrees Celsius while shaking. Following centrifugation check the protein concentration then divide the oxidized proteins into aliquots and store them under cold conditions.Prepare the denatured substrate by incubating 12 micromolar citrate synthase overnight in 40 millimolar HEPES and 4.5 molar guanidinium chloride. On the following day, open the fluorospectrometer software and go to time course measurement. Set the excitation bandwidth to 2.5 nanometers.The excitation wavelength to 360 nanometers. The emission wavelength to 360 nanometers. The emission bandwidth to five nanometers.And the data interval to 0.5 seconds. Set the temperature to 25 degrees Celsius. Prepare the sample by adding 1600 microliters of 40 millimolar HEPES to a quartz cuvette.Insert the cuvette into the sample holder of a fluorospectrometer allowing the sample to reach the desired temperature. After setting the stirring to 600 rpm begin the measurement until the baseline is established. To measure the citrate synthase aggregation in the absence of a chaperone, add 10 microliters of denatured citrate synthetase, 120 seconds into the measurement.Then continue the measurement for 1200 seconds. To measure the citrate synthase aggregation in the presence of Hsp33 add Hsp33 60 seconds into the measurement. After an additional 60 seconds add 10 microliters of denatured citrate synthetase and continue the measurement for 1200 seconds.Next, upload the data file to perform data analysis and noise removal by Kfits. To remove any noise choose analysis parameters use automatic best model and select noise is always above signal. Manually remove obvious outliers using the green and red adjustable lines which will filter the noises above the green line and below the red line.Following this, set the baseline and the fit curve. After applying the noise threshold, download the processed data. Prepare protein substrate complex samples by adding citrate synthetase to Hsp33 at a ratio of 1 to 1.5 at 43 degrees Celsius.Use at least four steps and incubate the samples for 15 minutes at 43 degrees Celsius after every addition to allow complex formation. Centrifuge the samples at 16000 times G at four degrees Celsius for 30 minutes to remove any aggregates. Then place the samples in 150 microliter glass inserts and transfer them into vials.Then place them in the proper trays. Hold buffers H and D at 25 degrees Celsius and hold the samples and quenching buffer at zero to two degrees Celsius. Next, manually turn on all cooling units of the mass spectrometer.Once all cooling systems reach their target temperatures manually switch on both the HPLC and loading pumps. Open the software which controls both pumps as well as the software which controls the mass spectrometer and make sure that the MS is on standby. Following this, disconnect the HPLC outlet valve from the MS source.Wash the system first with a triple cleaner solution. Then with buffer B and finally with buffer A.Then insert the pepsin column into the system. After washing the system with a steady flow and steady pressure, insert the HPLC outlet valve into the MS source and turn the MS on.Now open the software. Build a running sequence and enter all time points, sample names and locations in the trays as well as the buffer names and locations. After the run is complete analyze the peptide coverage of the sample by running the non-deuterated control samples through the software.Transfer data into the HDX Workbench software and analyze the results using the software. Save the selected peptides and continue further for analysis of the deuterium uptake. Chemical denaturation leads to a rapid citrate synthetase aggregation induced by the re-folding of a fully denaturized protein in unfavorable buffer conditions.On the other hand, the thermal-induced aggregation results from a relatively slow unfolding of the natively folded substrate. In both forms of denaturation, the addition of oxidized Hsp33 completely abolished aggregation, lowering the 360 nanometer readings to negligible values. The presence of a reduced Hsp33 had no effect on the citrate synthetase stability and gave an aggregation curve similar to the one detected in the absence of a chaperone.The deuteration pattern between the bound and unbound chaperone Hsp33 to its substrate citrate synthetase reveals a client interaction site within the C-terminal redox switch domain of the chaperone. Reduced Hsp33 has a completely ordered structure but becomes functional when it senses oxidative stress. While the reduced bound and unbound Hsp33 produces similar peptide curves the oxidized Hsp33 shows a 30%deuteration difference between the bound and unbound chaperone with specific areas of the bound protein indicating an allosteric hindrance.Once established the scattering assay can be completed within a few hours while the DHEX methodology can be completed in two to four days if performed properly. While attempting this procedure it is important to plan ahead. Remember that the condition described such as temperature, incubation times and buffer solutions are protein-specific and must be optimized for each protein.Following this procedure other methodologies such as cross-linking, or precipitation can be performed in order to complement studies of chaperone substrates interactions. In conclusion, this technique has paved the way for researchers in the field of structural biology to explore protein plasticity in molecular chaperones.

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