We thank Dr. Claudia Cremers for her helpful advice on chaperone assays. Ken Wan is acknowledged for his technical assistance in HdeB purification. This work was supported by the Howard Hughes Medical Institute (to J.C.A.B.) and the National Institutes of Health grant RO1 GM102829 to J.C.A.B. and U.J. J.-U. D. is supported by a postdoctoral research fellowship provided by the German Research Foundation (DFG).

1. Expression and Purification of Periplasmic HdeB
NOTE: HdeB was expressed in E. coli cells harboring the plasmid pTrc-hdeB10, and purified from the periplasm upon polymyxin lysis.
<ol>
	<li>Prepare an overnight culture of E. coli cells harboring the plasmid pTrc-hdeB 10 in 30 ml LB containing 200 &#181;g/ml ampicillin (LBAmp). Inoculate four 1 L cultures of LBAmp and grow them at 37 &#176;C and 200 rpm until O.D.600nm of 0.7 is reached. Then, add 300 &#181;M IPTG to induce the expression of HdeB and decrease the growth temperature to 30 &#176;C.</li>
	<li>After 5 hr of protein expression at 30 &#176;C, harvest the cells by centrifugation at 8,000 x g for 5 min at 4 &#176;C.</li>
	<li>Wash the cell pellet with 100 ml buffer A (50 mM Tris/HCl, 50 mM NaCl, pH 7.5) and centrifuge the cells again at 8,000 x g for 5 min at 4 &#176;C.</li>
	<li>Subsequently, resuspend the cell pellet in 80 ml buffer A, containing 1 mg/ml polymyxin sulfate. For efficient disruption of the outer membrane, gently stir the suspension for 1 hr at 4 &#176;C.</li>
	<li>To remove the cytoplasmic fraction and cell debris, centrifuge the suspension for 20 min at 15,000 x g at 4 &#176;C. This results in ~60 ml supernatant containing the soluble HdeB.</li>
	<li>Dialyze the supernatant containing the periplasmic extract overnight against 150x volume of buffer B (20 mM Tris/HCl, 0.5 mM EDTA, pH 8.0) using a dialysis membrane with 6 kDa MW cut-off. Concentrate the proteins to 15 ml using centrifugal filter units with a molecular weight cut-off of 3 kDa. Filter the protein solution using a 0.2 &#181;m pore filter.</li>
	<li>Apply the protein onto an anion exchange chromatography column (column volume 5 ml) that has been equilibrated with 5 column volumes buffer B with a flow rate of 2.5 ml/min. Once the protein is loaded onto the column, wash the column with buffer B for 10 min at a flow rate of 2.5 ml/min. Elute HdeB with a linear gradient from 0 to 0.5 M NaCl in buffer B over a time period of 50 min with a flow rate of 2.5 ml/min 6.</li>
	<li>Identify fractions containing HdeB using a 15% SDS-PAGE. Mix 20 &#181;l sample with 5 &#181;l 5x reduced SDS loading buffer. Load 10 &#181;l onto the gel and run in Tris-glycine buffer (14.4 g/L glycine, 2.9 g/L Tris, 1 g/L sodium dodecyl sulfate, pH 8.3). Run the gel at 150 V until the bromophenol band has migrated close to the bottom of the gel (~45 min).</li>
	<li>Pool all HdeB-containing fractions, dialyze at 4 &#176;C overnight against 4 L HdeB storage buffer (50 mM Tris/HCl, 200 mM NaCl, pH 8.0), and concentrate the protein to approximately 300 &#181;M using centrifugal filter units with a molecular weight cut-off of 3 kDa. Determine the concentration of HdeB at 280 nm using the extinction coefficient &#949;280nm= 15,595 M-1cm-1. Prepare 100 &#181;l aliquots and flash-freeze the aliquots in liquid nitrogen. 
	NOTE: HdeB can be stored at -70 &#176;C for at least 6 months.</li>
</ol>
2. Chaperone Activity Assay Using Thermally Unfolding Malate Dehydrogenase (MDH)
NOTE: The influence of purified HdeB on the aggregation of thermally unfolding porcine mitochondrial malate dehydrogenase (MDH) at different pH values was monitored as described below. All listed protein concentrations refer to the monomer concentration.
<ol>
	<li>To prepare MDH, dialyze MDH at 4 &#176;C overnight against 4 L buffer C (50 mM potassium phosphate, 50 mM NaCl, pH 7.5) and concentrate the protein to approximately 100 &#181;M using centrifugal filter units with a molecular weight cut-off of 30 kDa. 
	NOTE: Careful dialysis of MDH is required as MDH is delivered as ammonium sulfate solution.</li>
	<li>To remove aggregates, centrifuge the protein for 20 min at 20,000 x g at 4 &#176;C. Determine MDH concentration by absorbance at 280 nm (&#949;280 nm= 7,950 M-1 cm-1). Prepare 50 &#181;l aliquots of MDH and flash-freeze the aliquots for storage.</li>
	<li>Place 1 ml quartz cuvette into a fluorescence spectrophotometer equipped with temperature controlled sample holders and stirrer. Set &#955;ex/em to 350 nm.</li>
	<li>Add appropriate volumes of pre-warmed (43 &#176;C) buffer D (150 mM potassium phosphate, 150 mM NaCl) at the desired pH values (here: pH 2.0, pH 3.0, pH 4.0, and pH 5.0) to the cuvette and set the temperature in the cuvette holder to 43 &#176;C. The total volume is 1,000 &#181;l.</li>
	<li>Add 12.5 &#181;M HdeB (or alternatively the same volume of HdeB storage buffer for the buffer control) to the buffer, followed by the addition of 0.5 &#181;M MDH. Begin monitoring light scattering. Incubate the reaction for 360 sec to allow sufficient unfolding of MDH.</li>
	<li>Raise the pH to 7 by adding 0.16-0.34 volume of 2 M unbuffered K2HPO4 and continue recording light scattering for another 440 sec.</li>
	<li>Set the extent of MDH aggregation that is recorded in the absence of the chaperone at a defined time point after neutralization (here after 500 sec, when maximal light scattering of MDH was observed) to 100%. Normalize HdeB&#39;s activity to the light scattering signal of MDH in the absence of HdeB at each indicated pH value.</li>
</ol>
3. Detection of HdeB-LDH Complex Formation by Analytical Ultracentrifugation (aUC)
NOTE: Sedimentation velocity experiments of HdeB alone or in complex with thermally unfolding lactate dehydrogenase (LDH) were performed using an analytical ultracentrifuge.
<ol>
	<li>To prepare LDH, dialyze LDH at 4 &#176;C overnight against 4 L buffer C (50 mM potassium phosphate, 50 mM NaCl, pH 7.5) and concentrate the protein to approximately 200 &#181;M using centrifugal filter units with a molecular weight cut-off of 30 kDa. 
	NOTE: Careful dialysis of LDH is required as LDH is delivered as ammonium sulfate solution.</li>
	<li>To remove aggregates, centrifuge LDH for 20 min at 20,000 x g at 4 &#176;C. Determine LDH concentration by absorbance at 280 nm (&#949;280 nm= 43,680 M-1 cm-1). Prepare 50 &#181;l aliquots of LDH and flash-freeze the aliquots for storage.</li>
	<li>Incubate 3 &#181;M LDH in the presence and absence of 30 &#181;M HdeB in buffer D (pH 4 and 7, respectively) for 15 min at 41 &#176;C. 
	NOTE: Incubation of LDH at higher temperatures results in its complete aggregation, and no chaperone effect of HdeB can be observed.</li>
	<li>Let the samples cool down to room temperature. Then, load samples into cells containing standard sector shaped 2-channel centerpieces with 1.2 cm path length. Load the cells into the ultracentrifuge and equilibrate to 22 &#176;C for at least 1 hr prior to sedimentation.</li>
	<li>Spin samples at 22 &#176;C and 167,000 x g in the respective rotor for 12 hr, and monitor the sedimentation of the protein continuously at 280 nm. As previously demonstrated, the signal to noise ratio is improved when the transmitted light intensity of each channel is measured rather than absorbance. This also improves the quality of subsequent data fitting.</li>
	<li>Conduct data analysis with SEDFIT (version 15.01b, December 2015), using the continuous c(s) distribution model 17. A tutorial describing how to use SEDFIT can be found in reference 18.
	<ol>
		<li>Set the confidence level for the ME (Maximum Entropy) regularization to 0.7.</li>
	</ol>
	</li>
	<li>Calculate buffer density as well as viscosity using SEDNTERP 19. To estimate the amount of aggregated client protein, compare the integrals of sedimented LDH in pH 4 to pH 7 as a reference. 
	NOTE: Integration of the sedimentation distribution plots can be done directly in SEDFIT. Alternative software to analyze sedimentation velocity data can be found in a recent review 20.</li>
</ol>
4. Monitoring MDH Inactivation and Reactivation in the Presence of HdeB
NOTE: The influence of purified HdeB on the refolding of pH-unfolded MDH was determined by monitoring MDH activity upon neutralization.
<ol>
	<li>Incubate 1 &#181;M MDH in buffer D at the desired pH values (here: pH 2.0, pH 3.0, pH 4.0, and pH 5.0) for 1 hr at 37 &#176;C in the absence or presence of 25 &#181;M HdeB. Then, shift the temperature to 20&#176; C for 10 min. 
	NOTE: No MDH refolding was observed even in the presence of HdeB when MDH was incubated at temperatures higher than 37 &#176;C.</li>
	<li>To initiate refolding of acid denatured MDH, neutralize the samples to pH 7 by addition of 0.13-0.42 volume of 0.5 M sodium phosphate, pH 8.0.</li>
	<li>After incubation for 2 hr at 20 &#176;C, determine MDH activity by monitoring the decrease of NADH at 340 nm 9. 
	NOTE: MDH catalyzes the NADH-dependent reduction of oxaloacetate into L-malate.
	<ol>
		<li>Mix 50 &#181;l of the incubation reaction with 950 &#181;l of assay buffer (50 mM sodium phosphate, pH 8.0, 1 mM oxaloacetate, and 150 &#181;M NADH). 
		NOTE: The final concentration of MDH in the assay buffer should be 44 nM.</li>
		<li>Monitor the change in absorbance using a spectrophotometer, equipped with a Peltier temperature control block set to 20 &#176;C.</li>
		<li>Report the MDH activity relative to 44 nM native MDH that has been kept at pH 7.0.</li>
	</ol>
	</li>
</ol>
5. Effect of HdeB Overexpression on E. coli Survival under Acid Stress
NOTE: E. coli MG1655 genomic DNA was isolated using a published protocol 21.
<ol>
	<li>Amplify hdeB from E. coli MG1655 by PCR using primers hdeB-BamHI-rev GGT GGT CTG GGA TCC TTA ATT CGG CAA GTC ATT and hdeB-EcoRI-fw GGT GCC GAA TTC AGG AGG CGC ATG AAT ATT TCA TCT CTC C.</li>
	<li>Set up the PCR reaction in 50 &#181;l as follows: 10 &#181;l 5x polymerase buffer, 200 &#181;M dNTPs, 0.5 &#181;M primer JUD2, 0.5 &#181;M primer JUD5, 150 ng genomic DNA MG1655, 0.5 &#181;l DNA polymerase, add ddH2O to 50 &#181;l.</li>
	<li>Perform amplification of hdeB as follows: Step 1: 5 min at 95 &#176;C, 1 cycle; step 2: 30 sec at 95 &#176;C, 30 sec at 55 &#176;C, 30 sec at 72 &#176;C, 40 cycles; Step 3: 10 min at 72 &#176;C.</li>
	<li>Clone resulting PCR fragment into the EcoRI and BamHI sites of plasmid pBAD18 using standard methods for restriction site cloning. Purify the plasmid using a plasmid purification kit according to manufacturer&#39;s instructions. Verify the resulting plasmid by sequencing 10.</li>
	<li>Transform the plasmid expressing HdeB or the empty vector control pBAD18 into strain BB7224 (&#916;rpoH) (genotype: F-, &#955;-, e14-, [araD139]B/r &#916;(argF-lac)169 flhD5301 &#916;(fruK-yeiR)725(fruA25) relA1 rpsL150(SmR) rbsR22 &#916;(fimB-fimE)632(::IS1) ptsF25 zhf::Tn10(TcS) suhX401 deoC1 araD+ rpoH::kan+;16) using chemically competent cells. 
	NOTE: This strain is temperature-sensitive.</li>
	<li>After 45 sec heat-shock at 42 &#176;C and prior to the plating, incubate cells at 30 &#176;C and 200 rpm. Perform single colony streak-outs of the positive clones and incubate overnight at 30 &#176;C. Prepare an overnight culture in 50 ml LBAmp and cultivate the cells at 200 rpm and 30 &#176;C.</li>
	<li>Dilute overnight cultures 40-fold into 25 ml LBAmp and grow the bacteria in the presence of 0.5% arabinose (Ara) at 30 &#176;C and 200 rpm to an O.D.600nm = 1.0 to induce HdeB protein expression.</li>
	<li>For the pH shift experiments, use LBAmp+Ara to dilute the cells to O.D.600nm of 0.5 and adjust to the respective pH values (here: pH 2.0, pH 3.0, and pH 4.0) by adding appropriate volumes of 5 M HCl.</li>
	<li>After the indicated time points (pH 2, 1 min; pH 3, 2.5 min; pH 4, 30 min), neutralize the cultures by addition of the appropriate volumes of 5 M NaOH.</li>
	<li>Monitor the growth of the neutralized cultures in liquid culture for 12 hr at 30 &#176;C using O.D. measurements.</li>
</ol>

<ol>
	<li>Smith, 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=The+Role+of+Gastric+Acid+in+Preventing+Foodborne+Disease+and+How+Bacteria+Overcome+Acid+Conditions.">The Role of Gastric Acid in Preventing Foodborne Disease and How Bacteria Overcome Acid Conditions.</a> J Food Protect. 66, 1292-1303 (2003).</li><li>Hong, W., Wu, Y. E., Fu, X., Chang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperone-dependent+mechanisms+for+acid+resistance+in+enteric+bacteria.">Chaperone-dependent mechanisms for acid resistance in enteric bacteria.</a> Trends Microbiol. 20 (7), 328-335 (2012).</li><li>Koebnik, R., Locher, K. P., Van Gelder, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+bacterial+outer+membrane+proteins:+barrels+in+a+nutshell.">Structure and function of bacterial outer membrane proteins: barrels in a nutshell.</a> Mol Microbiol. 37, 239-253 (2000).</li><li>Reichmann, D., Xu, Y., 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=Order+out+of+Disorder:+Working+Cycle+of+an+Intrinsically+Unfolded+Chaperone.">Order out of Disorder: Working Cycle of an Intrinsically Unfolded Chaperone.</a> Cell. 148 (5), 947-957 (2012).</li><li>Bardwell, J. C. A., Jakob, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+disorder+in+chaperone+action.">Conditional disorder in chaperone action.</a> Trends Biochem Sci. 37 (12), 517-525 (2012).</li><li>Tapley, T. L., Korner, J. 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=Structural+plasticity+of+an+acid-activated+chaperone+allows+promiscuous+substrate+binding.">Structural plasticity of an acid-activated chaperone allows promiscuous substrate binding.</a> Proc Natl Acad Sci U S A. 106 (14), 5557-5562 (2009).</li><li>Hong, W., Jiao, W., 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=Periplasmic+Protein+HdeA+Exhibits+Chaperone-like+Activity+Exclusively+within+Stomach+pH+Range+by+Transforming+into+Disordered+Conformation.">Periplasmic Protein HdeA Exhibits Chaperone-like Activity Exclusively within Stomach pH Range by Transforming into Disordered Conformation.</a> J Biol Chem. 280 (29), 27029-27034 (2005).</li><li>Zhang, B. W., Brunetti, L., Brooks, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+pH-Dependent+Dissociation+of+HdeA+Dimers.">Probing pH-Dependent Dissociation of HdeA Dimers.</a> J Am Chem Soc. 133, 19393-19398 (2011).</li><li>Tapley, T. L., Franzmann, T. M., Chakraborty, S., Jakob, U., Bardwell, J. 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=Protein+refolding+by+pH-triggered+chaperone+binding+and+release.">Protein refolding by pH-triggered chaperone binding and release.</a> Proc Natl Acad Sci U S A. 107 (3), 1071-1076 (2010).</li><li>Dahl, J. -. U., Koldewey, P., Salmon, L., Horowitz, S., Bardwell, J. C. A., Jakob, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HdeB+Functions+as+an+Acid-protective+Chaperone+in+Bacteria.">HdeB Functions as an Acid-protective Chaperone in Bacteria.</a> J Biol Chem. 290 (1), 65-75 (2015).</li><li>Waterman, S. R., Small, P. L. C. <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+sigmas-dependent+genes+associated+with+the+stationary-phase+acid-resistance+phenotype+of+Shigella+flexneri.">Identification of sigmas-dependent genes associated with the stationary-phase acid-resistance phenotype of Shigella flexneri.</a> Mol Microbiol. 21 (5), 925-940 (1996).</li><li>Mucacic, M., Baneyx, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperone+Hsp31+Contributes+to+Acid+Resistance+in+Stationary-Phase+Escherichia+coli.">Chaperone Hsp31 Contributes to Acid Resistance in Stationary-Phase Escherichia coli.</a> Appl Environ Microbiol. 73 (3), 1014-1018 (2007).</li><li>Daugherty, D. L., Rozema, D., Hanson, P. E., Gellman, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+Chaperone-assisted+Refolding+of+Citrate+Synthase.">Artificial Chaperone-assisted Refolding of Citrate Synthase.</a> J Biol Chem. 273, 33961-33971 (1998).</li><li>Jakob, U., Muse, W., Eser, M., Bardwell, J. 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=Chaperone+Activity+with+a+Redox+Switch.">Chaperone Activity with a Redox Switch.</a> Cell. 96 (3), 341-352 (1999).</li><li>Guisbert, E., Yura, T., Rhodius, V. A., Gross, 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=Convergence+of+Molecular,+Modeling,+and+Systems+Approaches+for+an+Understanding+of+the+Escherichia+coli+Heat+Shock+Response.">Convergence of Molecular, Modeling, and Systems Approaches for an Understanding of the Escherichia coli Heat Shock Response.</a> Microbiol Mol Biol Rev. 72 (3), 545-554 (2008).</li><li>Tomoyasu, T., Mogk, A., Langen, H., Goloubinoff, P., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+the+roles+of+chaperones+and+proteases+in+protein+folding+and+degradation+in+the+Escherichia+coli+cytosol.">Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol.</a> Mol Microbiol. 40 (2), 397-413 (2001).</li><li>Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-Distribution+Analysis+of+Macromolecules+by+Sedimentation+Velocity+Ultracentrifugation+and+Lamm+Equation+Modeling.">Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling.</a> Biophys J. 78 (3), 1606-1619 (2000).</li><li>Patel, T. R., Winzor, D. J., Scott, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+ultracentrifugation:+A+versatile+tool+for+the+characterisation+of+macromolecular+complexes+in+solution.">Analytical ultracentrifugation: A versatile tool for the characterisation of macromolecular complexes in solution.</a> Methods. 95, 55-61 (2016).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Nucleic+Acids+by+Extraction+with+Phenol:Chloroform.">Purification of Nucleic Acids by Extraction with Phenol:Chloroform.</a> Cold Spring Harb Protoc. 2006, (2006).</li><li>Foit, L., George, J. S., Zhang, B. i. n. W., Brooks, C. L., Bardwell, J. 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=Chaperone+activation+by+unfolding.">Chaperone activation by unfolding.</a> Proc Natl Acad Sci U S A. 110, 1254-1262 (2013).</li><li>Nicoll, W. S., Boshoff, A., Ludewig, M. H., Hennessy, F., Jung, M., 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=Approaches+to+the+isolation+and+characterization+of+molecular+chaperones.">Approaches to the isolation and characterization of molecular chaperones.</a> Protein Express Purif. 46, 1-15 (2006).</li><li>Minami, Y., Hohfeld, J., Ohtsuka, K., Hartl, F. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+Heat-shock+Protein+70+Reaction+Cycle+by+the+Mammalian+DnaJ+Homolog,+Hsp40.">Regulation of the Heat-shock Protein 70 Reaction Cycle by the Mammalian DnaJ Homolog, Hsp40.</a> J Biol Chem. 271 (32), 19617-19624 (1996).</li><li>Quan, S., Koldewey, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+selection+designed+to+stabilize+proteins+uncovers+a+chaperone+called+Spy.">Genetic selection designed to stabilize proteins uncovers a chaperone called Spy.</a> Nat Struct Mol Biol. 18, 262-269 (2011).</li><li>Gray, M. J., Wholey, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphosphate+Is+a+Primordial+Chaperone.">Polyphosphate Is a Primordial Chaperone.</a> Mol Cell. 53 (5), 689-699 (2014).</li></ol>

The authors have nothing to disclose

In order to study the mechanism of activation and chaperone function of HdeB, large quantities of HdeB have to be expressed and purified. A number of expression vector systems are available for the production of high levels of a target protein, including pTrc or pBAD vectors, both of which were used in this study. The promoters are readily accessible for E. coli RNA polymerase and thus allow strongly upregulated expression of HdeB in any E. coli strain. This aspect is especially relevant for in vivo...

A common natural environment in which microbial pathogens experience acid-induced protein unfolding conditions is the mammalian stomach (pH range 1-4), whose acidic pH serves as an effective barrier against food-borne pathogens 1. Protein unfolding and aggregation, which is caused by amino acid side chain protonation, affects biological processes, damages cellular structures and eventually causes cell death 1,2. Since the pH of the bacterial periplasm equilibrates almost instantaneously with the environmental pH due to the free diffusion of protons through the porous outer membrane, periplasmic and inner membrane proteins of Gram-negative bacteria are the most vulnerable cellular components under acid-stress conditions 3. To protect their periplasmic proteome against rapid acid-mediated damage, Gram-negative bacteria utilize the acid-activated periplasmic chaperones HdeA and HdeB. HdeA is a conditionally disordered chaperone 4,5: At neutral pH, HdeA is present as a folded, chaperone-inactive dimer. Upon a pH shift below pH 3, HdeA&#39;s chaperone function is quickly activated 6,7. Activation of HdeA requires profound structural changes, including its dissociation into monomers, and the partial unfolding of the monomers 6-8. Once activated, HdeA binds to proteins that unfold under acidic conditions. It effectively prevents their aggregation both during the incubation at low pH as well as upon pH neutralization. Upon return to pH 7.0, HdeA facilitates the refolding of its client proteins in an ATP-independent manner and converts back into its dimeric, chaperone-inactive conformation 9. Similarly, the homologous chaperone HdeB is also chaperone-inactive at pH 7.0. Unlike HdeA, however, HdeB&#39;s chaperone activity reaches its apparent maximum at pH 4.0, conditions under which HdeB is still largely folded and dimeric 10. Moreover, further lowering the pH causes the inactivation of HdeB. These results suggest that despite their extensive homology, HdeA and HdeB differ in their mode of functional activation allowing them to cover a broad pH range with their protective chaperone function. One other chaperone that has been implicated in the acid resistance of E. coli is the cytoplasmic Hsp31, which appears to stabilize unfolded client proteins until neutral conditions are restored. The precise mode of Hsp31&#39;s action, however, has remained enigmatic 12. Given that other enteropathogenic bacteria such as Salmonella lack the hdeAB operon, it is very likely that other yet unidentified periplasmic chaperones might exist that are involved in acid resistance of these bacteria 11.
The protocols presented here allow to monitor the pH-dependent chaperone activity of HdeB in vitro and in vivo 10 and can be applied to investigate other chaperones such as Hsp31. Alternatively, the complex network of transcription factors that control the expression of hdeAB can potentially be investigated by the in vivo stress assay. To characterize the chaperone function of proteins in vivo, different experimental setups can be applied. One route is to apply protein unfolding stress conditions and phenotypically characterize mutant strains that either overexpress the gene of interest or carry a deletion of the gene. Proteomic studies can be conducted to identify which proteins no longer aggregate under stress conditions when the chaperone is present, or the influence of a chaperone on a specific enzyme can be determined during stress conditions using enzymatic assays 14-16. In this study, we chose to overexpress HdeB in an rpoH deletion strain, which lacks the heat shock sigma factor 32. RpoH controls the expression of all major E. coli chaperones and its deletion is known to increase sensitivity to environmental stress conditions that cause protein unfolding 15. The in vivo chaperone activity of HdeB was determined by monitoring its ability to suppress the pH sensitivity of the &#916;rpoH strain. Altogether, the protocols presented here provide a fast and straightforward approach to characterize the activity of an acid-activated chaperone in vitro as well as in the in vivo context.

<table><tbody><tr><td>NEB10-beta &lt;em&gt;E. coli&lt;/em&gt; cells</td><td>New England Biolabs</td><td>C3019I</td><td></td></tr><tr><td>Ampicillin</td><td>Gold Biotechnology</td><td>A-301-3</td><td></td></tr><tr><td>LB Broth mix, Lennox</td><td>LAB Express</td><td>3003</td><td></td></tr><tr><td>IPTG</td><td>Gold Biotechnology</td><td>I2481C50</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>S271-10</td><td></td></tr><tr><td>Tris</td><td>Amresco</td><td>0826-5kg</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP120-500</td><td></td></tr><tr><td>Polymyxin B sulfate&amp;nbsp;</td><td>ICN Biomedicals Inc.</td><td>100565</td><td></td></tr><tr><td>0.2 &amp;micro;m pore sterile Syringe Filter</td><td>Corning</td><td>431218</td><td></td></tr><tr><td>HiTrap Q HP (CV 5 ml)</td><td>GE Healthcare Life Sciences</td><td>17-1153-01</td><td></td></tr><tr><td>Mini-Protean TGX, 15%</td><td>Bio-Rad</td><td>4561046</td><td></td></tr><tr><td>Malate dehydrogenase (MDH)</td><td>Roche</td><td>10127914001</td><td></td></tr><tr><td>Potassium phosphate (Monobasic)</td><td>Fisher Scientific</td><td>BP362-500</td><td></td></tr><tr><td>Potassium phosphate (Dibasic)</td><td>Fisher Scientific</td><td>BP363-1</td><td></td></tr><tr><td>F-4500 fluorescence spectrophotometer</td><td>Hitachi</td><td>FL25</td><td></td></tr><tr><td>Oxaloacetate</td><td>Sigma</td><td>O4126-5G</td><td></td></tr><tr><td>NADH</td><td>Sigma&amp;nbsp;</td><td>N8129-100MG</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma&amp;nbsp;</td><td>S9390-2.5KG</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma&amp;nbsp;</td><td>S397-500</td><td></td></tr><tr><td>Lactate dehydrogenase (LDH)</td><td>Roche</td><td>10127230001</td><td></td></tr><tr><td>Beckman Proteome Lab XL-I analytical Ultracentrifuge</td><td>Beckman Coulter</td><td>&lt;a href=&#039;https://www.beckmancoulter.com/wsrportal/wsrportal.portal?_nfpb=true&amp;amp;_windowLabel=UCM_RENDERER&amp;amp;_urlType=render&amp;amp;wlpUCM_RENDERER_path=%252Fwsr%252Fresearch-and-discovery%252Fproducts-and-services%252Fcentrifugation%252Fproteomelab-xl-a-xl-i%252Findex.htm#2/10//0/25/1/0/asc/2/392764///0/1//0/%2Fwsrportal%2Fwsr%2Fresearch-and-discovery%2Fproducts-and-services%2Fcentrifugation%2Fproteomelab-xl-a-xl-i%2Findex.htm/&#039;&gt;392764&lt;/a&gt;</td><td></td></tr><tr><td>Centerpiece, 12 mm, Epon Charcoal-filled</td><td>Beckman Coulter</td><td>306493</td><td></td></tr><tr><td>AN-50 Ti Rotor, Analytical, 8-Place</td><td>Beckman Coulter</td><td>363782</td><td></td></tr><tr><td>Wizard Plus Miniprep Kit</td><td>Promega</td><td>A1470</td><td>used for plasmid purification (Protocol 5.1)</td></tr><tr><td>L-arabinose</td><td>Gold Biotechnology</td><td>A-300-500</td><td></td></tr><tr><td>Glycine</td><td>DOT Scientific Inc</td><td>DSG36050-1000</td><td></td></tr><tr><td>Fluorescence Cell cuvette</td><td>Hellma Analytics</td><td>119004F-10-40</td><td></td></tr><tr><td>Oligonucleotides</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Phusion High-Fidelity DNA polymerase</td><td>New England Biolabs</td><td>M0530S</td><td></td></tr><tr><td>dNTP set</td><td>Invitrogen</td><td>10297018</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher Scientific</td><td>A144-212</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>BP359-500</td><td></td></tr><tr><td>Amicon Ultra 15 ml 3K NMWL</td><td>Millipore</td><td>UFC900324</td><td></td></tr><tr><td>Centrifuge Avanti J-26XPI</td><td>Beckman Coulter</td><td>393127</td><td></td></tr><tr><td>Varian Cary 50 spectrophotometer</td><td>Agilent Tech</td><td></td><td></td></tr><tr><td>Spectra/Por 1 Dialysis Membrane MWCO: 6 kDa</td><td>Spectrum Laboratories</td><td>132650</td><td></td></tr><tr><td>Amicon Ultra Centrifugal Filter Units 30K</td><td>Millipore</td><td>UFC803024</td><td></td></tr><tr><td>SDS</td><td>Fisher Scientific</td><td>bp166-500</td><td></td></tr><tr><td>Veriti 96-Well Thermal Cycler</td><td>Thermo Fisher</td><td>4375786</td><td></td></tr></tbody></table>

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.

HdeA and HdeB are homologous E. coli proteins, known to protect periplasmic proteins against acid stress conditions 10. Our work revealed that similar to HdeA, HdeB also functions as an acid activated molecular chaperone. However, in contrast to HdeA, HdeB functions at a pH that is still potentially bactericidal, but significantly higher than the pH optimum of HdeA 6,9,10,22. To investigate the pH optimum of HdeB&#39;s chaperone activity in vitro, n...

Watch this Scientific Journal Video about Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo at JoVE.com

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.

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 ...

detection-ph-dependent-activity-escherichia-coli-chaperone-hdeb-vitro

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

10.3K Views.  University of Michigan. 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.

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

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

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 ...

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 ...

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. Biomolecules such as proteins, carbohydrates, vitamins, fatty acids, nucleic acids, and enzymes are fundamental building blocks of living beings; they assemble into complex networks in biosystems to synchronize a myriad of life events. Proteins typically utilize complex interactome networks to carry out their functions; hence it is mandatory to evaluate such interactions to unravel their importance in cells at both cellular and organism levels. Toward this goal, we introduce a rapidly emerging technology, field-effect biosensing (FEB), to determine specific biomolecular interactions. FEB is a benchtop, label-free, and reliable biomolecular detection technique to determine specific interactions and uses high-quality electronic-based biosensors. The FEB technology can monitor interactions in the nanomolar range due to the biocompatible nanomaterials used on its biosensor surface. As a proof of concept, the protein-protein interaction (PPI) between heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) was elucidated. Hsp90 is an ATP-dependent molecular chaperone that plays an essential role in the folding, stability, maturation, and quality control of many proteins, thereby regulating multiple vital cellular functions. Cdc37 is regarded as a protein kinase-specific molecular chaperone, as it specifically recognizes and recruits protein kinases to Hsp90 to regulate their downstream signal transduction pathways. As such, Cdc37 is considered a co-chaperone of Hsp90. The chaperone-kinase pathway (Hsp90/Cdc37 complex) is hyper-activated in multiple malignancies promoting cellular growth; therefore, it is a potential target for cancer therapy. The present study demonstrates the efficiency of FEB technology using the Hsp90/Cdc37 model system. FEB detected a strong PPI between the two proteins (KD values of 0.014 &#181;M, 0.053 &#181;M, and 0.072 &#181;M in three independent experiments). In summary, FEB is a label-free and cost-effective PPI detection platform, which offers fast and accurate measurements.

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. ...

Assessment of Hsp70 Chaperone Activity Using Escherichia coli dnaK756 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. 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.

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.

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

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

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

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

Summary

Explore More Videos

Chapters in this video

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

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

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

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

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

Intracellular Refolding Assay

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions