The work was supported with grant funding obtained from the International Centre for Genetic Engineering and Biotechnology (ICGEB) grant number, HDI/CRP/012, Research Directorate of the University of Venda, grant I595, Department of Science and Innovation (DSI) and the National Research Foundation (NRF) of South Africa (grant numbers, 75464 &#38; 92598) awarded to AS.

1. Transformation
NOTE: Use sterile glassware for culture, pipette tips, and freshly prepared and autoclaved media. Prepare cultures of the E. coli cells in 2x yeast tryptone (YT) [1.6% tryptone (w/v), 1% yeast extract (w/v), 0.5% NaCl (w/v), 1.5% agar (w/v)] agar. General reagents used in the protocol and their sources are provided in the Table of Materials.
<ol>
	<li>Label 2.0 mL microcentrifuge tubes and aliquot 50 &#181;L of competent E. coli dnaK756cells, keeping the cells on ice.</li>
	<li>To the 2.0 mL microcentrifuge tubes with competent cells, aliquot 10-50 ng of pQE60/DnaK, pQE60/KPf, and pQE60/KPf-V436F plasmid DNA9&#160;into separate tubes.</li>
	<li>Keep the 2.0 mL microcentrifuge tubes containing the competent cells and plasmid DNA on ice for 30 min.</li>
	<li>Heat shock the competent cells-DNA mix for 60 s at 42 &#176;C and return the microcentrifuge tubes back on ice for 10 min.</li>
	<li>Add 950 &#181;L of fresh 2x YT broth (pre-incubated at 37 &#176;C) and incubate at 37 &#176;C while shaking at 150 revolutions/min for 1 h. Leave the cells to grow for much longer to encourage their recovery if necessary. 
	NOTE: Avoid shaking the cells vigorously.</li>
	<li>Pipette 100 &#181;L of the cells and spread them onto 2x YT agar plates containing 50 &#181;g/mL kanamycin, 10 &#181;g/mL tetracycline for the respective strain, and 100 &#181;g/mL ampicillin (see Table of Materials) for plasmid selection.</li>
	<li>Centrifuge the rest of the cells (whose volume is now approximately 900 &#181;L) for 1 min at 5000 x g (at 4 &#176;C) using a benchtop microcentrifuge.</li>
	<li>Decant about 800 &#181;L of the broth and use the remaining medium to resuspend the pelleted cells.</li>
	<li>Plate the recovered cells onto the 2x YT agar plate.</li>
	<li>Incubate both agar plates overnight (or approximately 17 h) at 37 &#176;C. 
	NOTE: These cells grow very slowly and may need to be incubated for much longer. Be careful to spot the colonies which may start off very small. The plate containing cells resuspended after centrifugation (step 1.7) serves as a back-up in case the transformation efficiency of the cells is poor, in which case the pelleted cells may improve the recovery of any transformed cells. However, if the transformation efficiency is excellent, the agar plate onto which concentrated cells were plated may be characterized by an overgrown culture upon incubation, making it difficult to identify single colonies. In that case, the other agar plate may be the one on which well-spaced colonies grow.</li>
</ol>
2. Cell plating
<ol>
	<li>Pick up a single colony from the transformants and inoculate it into 10 mL of 2x YT broth supplemented with 50 &#181;g/mL of kanamycin, 10 &#181;g/mL of tetracycline for the selection of the E. coli dnaK756&#160;cells, and 100 &#181;g/mL ampicillin for plasmid selection. 
	NOTE: Use flasks (&#8805;50 mL) to ensure aeration when agitating the culture. Incubate the inoculum overnight (17 h) at 37 &#176;C while shaking at 150 rotations/min.</li>
	<li>Take an absorbance reading at OD600&#160;the following morning.</li>
	<li>Clean the workbench surface using 75% ethanol to swab the surface in preparation for cell spotting.</li>
	<li>Using a 2 mL microcentrifuge tube, standardize the culture to an OD600&#160;reading of 2.0 using 2x YT broth. 
	NOTE: Ensure that the OD readings are taken correctly as this step is important for standardizing cell density across the various samples.</li>
	<li>Using 2 mL microcentrifuge tubes, prepare serial dilutions of the cells from 100&#160;to 10-5.</li>
	<li>Incubate the agar plates to be used to spot the cells onto in an oven set at 40 &#176;C to allow the plates to dry with lids partially open to ensure water vapor escapes. 
	NOTE: To ensure the cells are spotted at uniformly separated distances, draw lines on a piece of paper onto which a template of spotting sites is printed.</li>
	<li>Spot 2 &#181;L of the serially diluted cells onto the agar plates supplemented with 50 &#181;g/mL kanamycin, 10 &#181;g/mL tetracycline, 100 &#181;g/mL ampicillin, and 0.5 mM IPTG (for the induction of expression of the recombinant proteins) (see Table of Materials).</li>
	<li>Spot each sample onto two separate plates (one to be incubated at 37 &#176;C and the other at 43.5 &#176;C). 
	NOTE: Avoid piercing the agar plate.</li>
	<li>Spot control samples onto the same plate as the experimental samples to minimize environmental effects.</li>
	<li>Conduct the spotting quickly to ensure the process is completed before the cells start growing, as this may generate skewed growth patterns. 
	NOTE: It is important to avoid aerosols when spotting, as these would contaminate the plates. It is also important to keep the plates closed in between the spotting to avoid contamination.</li>
	<li>Incubate one plate at 37 &#176;C (permissive growth temperature) and the other at the non-permissive growth temperature of 43.5 &#176;C. 
	NOTE: Incubate the plates facing upside down to avoid steam collecting onto the lid, as the condensed water would wash the spotted colonies off their positions.</li>
	<li>Place all the plates in the incubators at the same time and avoid opening the incubator until the following morning. 
	NOTE: Opening the incubator door several times during incubation of the plates is discouraged. This is because the access of air into the incubator may result in temperature fluctuations that adversely impact cell growth.</li>
</ol>
3. Confirming expression of recombinant proteins
<ol>
	<li>Using a sterile loop, pick up part of the remaining cells from the same colony of transformed E. coli dnaK756&#160;cells.</li>
	<li>Inoculate the cells into 10 mL YT broth supplemented with 50 &#181;g/mL kanamycin, 10 &#181;g/mL tetracycline, and 100 &#181;g/mL ampicillin. Incubate overnight (17 h) at 37 &#176;C while shaking at 150 revolutions/min.</li>
	<li>Transfer the culture into 90 mL of sterile 2x YT broth containing the necessary antibiotics as mentioned in step 3.2. Let the cells grow to mid-log phase (OD600 = 0.4-0.6).</li>
	<li>Take 2 mL of the sample culture before induction (0 h induction sample).</li>
	<li>Add IPTG to a final concentration of 1 mM to induce protein production and re-incubate the cells at 37 &#176;C.</li>
	<li>Take a second 2 mL sample 6 h post-induction.</li>
	<li>Harvest the cells by centrifuging at 5000 x g for 10 min. Keep the centrifuge temperature at 4 &#176;C.</li>
	<li>Discard the supernatant.</li>
	<li>Resuspend the pellet in PBS buffer (137 mM NaCl, 27 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4) and store at -20 &#176;C.</li>
</ol>
4. SDS-PAGE and western blot analyses
<ol>
	<li>Prepare 2x 10% SDS gels as previously described19.</li>
	<li>Keep one of the SDS-PAGE gels for subsequent staining using Coomassie stain to view the protein bands. Use the second SDS-PAGE gel to conduct western blot analysis.</li>
	<li>Aliquot 80 &#181;L of the resuspended samples and mix it with 20 &#181;L of 4x Laemmli SDS loading buffer.</li>
	<li>Boil the suspension at 100 &#176;C for 10 min. Then load 10 &#181;L of each sample onto the precast SDS gel (see Table of Materials).</li>
	<li>Perform electrophoresis at room temperature for 1 h using a voltage of 120 V in a 1x solution of SDS running buffer (25 mm Tris, 250 mm glycine, 0.1% (w/v) SDS).</li>
	<li>Stain the gel with Coomassie stain (see Table of Materials) for 1 h, followed by destaining using destaining buffer (50% (v/v) methanol, 10% (v/v) acetic acid in distilled water) for 2 h.</li>
	<li>Visualize the protein bands present in the gel using a gel imaging system (see Table of Materials). Rerun another SDS-PAGE gel with the same samples following the above-mentioned protocol.</li>
	<li>When the electrophoretic run ends, take SDS-PAGE gels to conduct western blot analysis as previously described19.</li>
	<li>Wash the nitrocellulose membrane onto which the proteins were transferred three times in wash buffer (TBS-Tween, pH 7.4 [50 mM Tris, 150 mM NaCl, 1% (v/v) Tween]).</li>
	<li>Use &#945;-DnaK (see Table of Materials) to detect DnaK and &#945;-PfHsp70-17&#160;to detect KPf and its mutant, KPf-V436F), respectively.</li>
	<li>Use the antibodies at a 1:2000 dilution in 5% non-fat milk. Incubate at 4 &#176;C while shaking at 60 revolutions/min for 1 h.</li>
	<li>Remove non-specifically bound antibody by washing the nitrocellulose membrane in TBS-Tween 3 times in 15 min.</li>
	<li>Incubate the membrane in the secondary antibody (&#945;-rabbit, see Table of Materials) under the same conditions as the previous step. This is followed by subsequent washing under the same conditions as the primary antibody.</li>
	<li>Resolve the bands using enhanced chemiluminescence (ECL) detection reagent.</li>
	<li>Visualize the bands using a gel imager.</li>
</ol>

Assessment of Hsp70 Chaperone Activity Using Escherichia coli dnaK756 Cells

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Immunol. 58 (1), 61-74 (2010).</li><li>Mogk, 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=Identification+of+thermolabile+Escherichia+coli+proteins:+prevention+and+reversion+of+aggregation+by+DnaK+and+ClpB.">Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB.</a> EMBO J. 18 (24), 6934-6949 (1999).</li><li>Edkins, A. L., Boshoff, A., Shonhai, A., Picard, D., Blatch, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+structural+and+functional+features+of+molecular+chaperones.+Heat+shock+proteins+of+malaria.">General structural and functional features of molecular chaperones. Heat shock proteins of malaria.</a> Adv Exp Med Biol. , (2021).</li><li>Bertelsen, E. B., Chang, L., Gestwicki, J. E., Zuiderweg, E. 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The authors have no competing financial interests or other conflicts of interest.

The protocol demonstrates the utility of E. coli dnaK756cells in exploring the chaperone function of heterologously expressed Hsp70. This assay could be adopted to screen inhibitors targeting Hsp70 function in cellulo. However, one limitation of this method is that Hsp70s unable to substitute for DnaK in E. coli are not compatible with this assay. Lack of post-translational modification21&#160;of some non-native Hsp70s may account for their lack of function within the <e...

Heat shock proteins play an important role as molecular chaperones by facilitating protein folding, preventing protein aggregation, and reversing protein misfolding1,2. Heat shock protein 70 (Hsp70) is one of the most prominent molecular chaperones, playing a central role in protein homeostasis3,4. DnaK is the E. coli Hsp70 homologue5.
Various biophysical, biochemical, and cell-based assays have been developed to explore the chaperone activity of Hsp70 and to screen for inhibitors targeting this chaperone6,7,8. Hsp70 is a highly conserved protein. For this reason, several Hsp70s of eukaryotic organisms, such as Plasmodium falciparum (the main agent of malaria), have been reported to substitute for DnaK function in E. coli6,9. In this way, an E. coli-based complementation assay has been developed involving the heterologous expression of Hsp70s in E. coli to explore their cytoprotective function. Typically, this assay involves the utilization of E. coli cells that are either deficient for DnaK or that express a native DnaK that is functionally compromised. While DnaK is not essential for E. coli growth under normal conditions, it becomes essential when the cells are grown under stressful conditions such as elevated temperatures or other forms of stress10,11.
E. coli strains that have been developed to study Hsp70 function using a complementation assay include E. coli dnaK103&#160;(BB2393 [C600 dnaK103(Am) thr::Tn10]) and E. coli dnaK756. E. coli dnaK103&#160;cells produce a truncated DnaK that is non-functional, and as such, the cells grow adequately at 30 &#176;C, while the strain is sensitive to cold and heat stress12,13. Similarly, the E. coli dnaK756/BB2362 (dnaK756&#160;recA::TcR Pdm1,1) strain does not grow above 40 &#176;C14,15. The E. coli dnaK756&#160;strain expresses a mutant native DnaK (DnaK756) characterized by three glycine-to-aspartate substitutions at positions 32, 455, and 468, giving rise to compromised proteostatic outcomes. Consequently, this strain is resistant to bacteriophage &#955; DNA14. Additionally, E. coli&#160;dnaK756 exhibits elevated ATPase activity, while its affinity for the nucleotide exchange factor, GrpE, is reduced16. E. coli DnaK mutant strains serve as ideal models for investigating the chaperone activity of Hsp70 through a complementation approach. Since DnaK is only essential under stressful conditions, the complementation assay is typically conducted at elevated temperatures (Figure 1). Some advantages of using E. coli for this study include its well-characterized genome, rapid growth, and the low cost of culturing and maintenance17.
In this article, we describe in detail a protocol involving the use of E. coli dnaK756&#160;cells to study the function of Hsp70. The Hsp70s we employed in the assay are wild-type DnaK and its chimeric derivative, KPf (made up of the ATPase domain of DnaK fused to the C-terminal substrate-binding domain of Plasmodium falciparum Hsp70-16,18). KPf-V436F was heterologously expressed as a negative control since the mutation essentially blocks it from binding substrates, thus abrogating its chaperone activity9.

<table><tbody><tr><td>2-&amp;beta;-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>8,05,740</td><td>Constituent for sample loading dye</td></tr><tr><td>Acetic acid</td><td>Labchem</td><td>101005125</td><td>Constituent of destainer</td></tr><tr><td>Acrylamide</td><td>Sigma-Aldrich</td><td>8008300100</td><td>Component of SDS</td></tr><tr><td>Agar</td><td>Merck</td><td>HG000BX1.500</td><td>Constituent of medium and liquid growth assay</td></tr><tr><td>Agarose</td><td>Clever Scientific</td><td>14131031</td><td>Certified molecular biology agarose</td></tr><tr><td>Ammonium persulfate</td><td>Sigma-Aldrich</td><td>101875295</td><td>Constituent for SDS-PAGE gel</td></tr><tr><td>Ampicillin</td><td>VWR International</td><td>0339&amp;mdash;EU&amp;mdash;25G</td><td>Selective antibiotic</td></tr><tr><td>Bis</td><td>Sigma-aldrich</td><td>1015460100</td><td>Component of SDS</td></tr><tr><td>Bromophenol</td><td>Sigma-Aldrich</td><td>0449-25G</td><td>Constituent for sample loading dye</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>10043-52-4</td><td>For competent cells preparation</td></tr><tr><td>Coomassie brilliant blue</td><td>VWR International</td><td>443293X</td><td>SDS-PAGE dye</td></tr><tr><td>Dibasic sodium phosphate</td><td>Sigma-Aldrich</td><td>RB10368</td><td>Constituent of PBS buffer</td></tr><tr><td>ECL</td><td>Thermofischer Scientific</td><td>32109</td><td>Western blot detection reagent</td></tr><tr><td>Ethidium Bromide</td><td>Thermofischer Scientific</td><td>17898</td><td>DNA intercalating dye</td></tr><tr><td>Glycerol</td><td>Merck</td><td>SAAR2676520L</td><td>Constituent for sample loading dye</td></tr><tr><td>Glycine</td><td>VWR International</td><td>10119CU</td><td>Component of SDS</td></tr><tr><td>IPTG</td><td>Glentham life sciences</td><td>162IL</td><td>inducer</td></tr><tr><td>Kanamycin</td><td>Melford</td><td>K0126</td><td>Selective antibiotic</td></tr><tr><td>Magnesium Chloride</td><td>Merck</td><td>SAAR4123000EM</td><td>Constituent of medium and liquid growth assay</td></tr><tr><td>Methanol</td><td>Labchem</td><td>113140129</td><td>Constituent of destainer</td></tr><tr><td>Monobasic potassium phosphate</td><td>Merck</td><td>1,04,87,30,250</td><td>Constituent of PBS buffer</td></tr><tr><td>Peptone</td><td>Merck</td><td>HG000BX4.250</td><td>Constituent of medium and liquid growth assay</td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>SAAR5042020EM</td><td>Constituent of PBS buffer</td></tr><tr><td>PVDF membrane</td><td>Thermofischer scientific</td><td>PB7320</td><td>Western blot membrane</td></tr><tr><td>Sodium Chloride</td><td>Merck</td><td>SAAR5822320EM</td><td>Constituent of medium and liquid growth assay</td></tr><tr><td>Sodium dodecyl sulphate</td><td>VWR International</td><td>108073</td><td>To resolve expressed proteins</td></tr><tr><td>Spectramax iD3</td><td>Separations</td><td>373705019</td><td>Automated plate reader</td></tr><tr><td>TEMED</td><td>VWR international</td><td>ACRO420580500</td><td>Component of SDS gel</td></tr><tr><td>Tetracycline</td><td>Duchefa Biochemies</td><td>T0150.0025</td><td>Selective antibiotic</td></tr><tr><td>Tris</td><td>VWR International</td><td>19A094101</td><td>Component of SDS gel</td></tr><tr><td>Tween20</td><td>Merck</td><td>SAAR3164500XF</td><td>Constituent for Western wash buffer</td></tr><tr><td>Western transfer chamber</td><td>Thermofisher Scientific</td><td>PB0112</td><td>Transfer of protein to nitrocellulose membrane</td></tr><tr><td>Yeast extract</td><td>Merck</td><td>HG000BX6.500</td><td>Constituent of medium and liquid growth assay</td></tr><tr><td>&amp;alpha;-DnaK antibody</td><td>Inqaba</td><td>BK CAC09317</td><td>Primary antibody</td></tr><tr><td>&amp;alpha;-rabbit antibody</td><td>Thermofischer scientific</td><td>31460</td><td>Secondary antibody</td></tr></tbody></table>

escherichia coli -based complementation assay to study the chaperone function of heat shock protein 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. While Hsp70 is not essential for E. coli cells growing under normal conditions, this chaperone becomes indispensable for growth at elevated temperatures. Since Hsp70 is highly conserved, one way to study the chaperone function of Hsp70&#160;genes from various species is to heterologously express them in E. coli strains that are either deficient in Hsp70 or express a native Hsp70 that is functionally compromised. E. coli dnaK756&#160;cells are unable to support &#955; bacteriophage DNA. Furthermore, their native Hsp70 (DnaK) exhibits elevated ATPase activity while demonstrating reduced affinity for GrpE (Hsp70 nucleotide exchange factor). As a result, E. coli dnaK756&#160;cells grow adequately at temperatures ranging from 30 &#176;C to 37 &#176;C, but they die at elevated temperatures (&#62;40 &#176;C). For this reason, these cells serve as a model for studying the chaperone activity of Hsp70. Here, we describe a detailed protocol for the application of these cells to conduct a complementation assay, enabling the study of the in cellulo chaperone function of Hsp70.

Figure 2&#160;presents an image of the scanned agar containing cells that were spotted and cultured at the permissive growth temperature of 37 &#176;C and 43.5 &#176;C, respectively. On the right-hand side of Figure 2, excised western blot components represent the expression of DnaK, KPf, and KPf-V436F in E. coli dnaK756&#160;cells. As expected, all the E. coli dnaK756&#160;cells cultured at the permissive growth temperature of 37 &#176;C manag...

Watch this Scientific Journal Video about Author Spotlight: Exploring Heat Shock Proteins in Malaria and Tuberculosis Infections at JoVE.com

Author Spotlight: Exploring Heat Shock Proteins in Malaria and Tuberculosis Infections

This protocol demonstrates the chaperone activity of heat shock protein 70 (Hsp70). E. coli dnaK756 cells serve as a model for the assay as they harbor a native, functionally impaired Hsp70, making them susceptible to heat stress. The heterologous introduction of functional Hsp70 rescues the growth deficiency of the cells.

Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. ...

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Biochemistry

1.1K Views.  University of Venda. This protocol demonstrates the chaperone activity of heat shock protein 70 (Hsp70). E. coli dnaK756 cells serve as a model for the assay as they harbor a native, functionally impaired Hsp70, making them susceptible to heat stress. The heterologous introduction of functional Hsp70 rescues the growth deficiency of the cells.

Video: Author Spotlight: Exploring Heat Shock Proteins in Malaria and Tuberculosis Infections

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

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.

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 ...

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level. While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is&#160;presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay. Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Here, standardized protocols are presented to assess induction of the heat shock response (HSR) in Caenorhabditis elegans using RT-qPCR at the molecular level, fluorescent reporters at the cellular level, and thermorecovery at the organismal level.

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, ...

Environment

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...

Intracellular Refolding Assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45&deg;C for 30 minutes. Upon recovery at 37&deg;C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

Biology

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell cytoskeletal structures. This involves the assembly-disassembly of higher-order macromolecular structures at a given time and location, a process that is particularly sensitive to perturbations caused by overexpression of proteins. Methods that can preserve protein homeostasis and maintain near-to-normal cellular morphology are highly desirable to determine the functional contribution of a protein of interest in a wide range of cellular processes. Transient depletion-rescue experiments based on RNA interference are powerful approaches to analyze protein functions and structural requirements. However, reintroduction of the target protein with minimum deviation from its physiological level is a real challenge. Here we describe a method termed adenofection that was developed to study the role of molecular chaperones and partners in the normal operation of dividing cells and the relationship with actin remodeling. HeLa cells were depleted of BAG3 with siRNA duplexes targeting the 3&#39;UTR region. GFP-tagged BAG3 proteins were reintroduced simultaneously into &#62;75% of the cells using recombinant adenoviruses coupled to transfection reagents. Adenofection enabled to express BAG3-GFP proteins at near physiological levels in HeLa cells depleted of BAG3, in the absence of a stress response. No effect was observed on the levels of endogenous Heat Shock Protein chaperones, the main stress-inducible regulators of protein homeostasis. Furthermore, by adding baculoviruses driving the expression of fluorescent markers at the time of cell transduction-transfection, we could dissect mitotic cell dynamics by time-lapse microscopic analyses with minimum perturbation of normal mitotic progression. Adenofection is applicable also to hard-to-infect mouse cells, and suitable for functional analyses of myoblast differentiation into myotubes. Thus adenofection provides a versatile method to perform structure-function analyses of proteins involved in sensitive biological processes that rely on higher-order cytoskeletal dynamics.

We describe a method for depletion-rescue experiments that preserves cellular integrity and protein homeostasis. Adenofection enables functional analyses of proteins within biological processes that rely on finely tuned actin-based dynamics, such as mitotic cell division and myogenesis, at the single-cell level.

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70

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Chapters in this video

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

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

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Intracellular Refolding Assay

A Protocol for the Identification of Protein-protein Interactions Based on ¹⁵N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging