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>

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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.

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			<td height="63" style="height:63px;width:216px;">NEB10-beta E. coli cells</td>
			<td style="width:64px;">New England Biolabs</td>
			<td style="width:119px;">C3019I</td>
			<td style="width:1791px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ampicillin</td>
			<td style="width:64px;">Gold Biotechnology</td>
			<td style="width:119px;">A-301-3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LB Broth mix, Lennox</td>
			<td style="width:64px;">LAB Express</td>
			<td style="width:119px;">3003</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">IPTG</td>
			<td style="width:64px;">Gold Biotechnology</td>
			<td style="width:119px;">I2481C50</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sodium chloride</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">S271-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:64px;">Amresco</td>
			<td style="width:119px;">0826-5kg</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">EDTA</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">BP120-500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Polymyxin B sulfate&#160;</td>
			<td style="width:64px;">ICN Biomedicals Inc.</td>
			<td style="width:119px;">100565</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0.2 UM pore sterile Syringe Filter</td>
			<td style="width:64px;">Corning</td>
			<td style="width:119px;">431218</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">HiTrap Q HP (CV 5 ml)</td>
			<td style="width:64px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-1153-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mini-Protean TGX, 15%</td>
			<td style="width:64px;">Bio-Rad</td>
			<td style="width:119px;">4561046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Malate dehydrogenase (MDH)</td>
			<td style="width:64px;">Roche</td>
			<td style="width:119px;">10127914001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Potassium phosphate (Monobasic)</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">BP362-500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Potassium phosphate (Dibasic)</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">BP363-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">F-4500 fluorescence spectrophotometer</td>
			<td style="width:64px;">Hitachi</td>
			<td style="width:119px;">FL25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxaloacetate</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:119px;">O4126-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NADH</td>
			<td style="width:64px;">Sigma&#160;</td>
			<td style="width:119px;">N8129-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium phosphate monobasic</td>
			<td style="width:64px;">Sigma&#160;</td>
			<td style="width:119px;">S9390-2.5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium phosphate dibasic</td>
			<td style="width:64px;">Sigma&#160;</td>
			<td style="width:119px;">S397-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lactate dehydrogenase (LDH)</td>
			<td style="width:64px;">Roche</td>
			<td style="width:119px;">10127230001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Beckman Proteome Lab XL-I analytical Ultracentrifuge</td>
			<td style="width:64px;">Beckman Coulter</td>
			<td style="width:119px;">392764</td>
			<td>https://www.beckmancoulter.com/wsrportal/wsrportal.portal?_nfpb=true&#38;_windowLabel=UCM_RENDERER&#38;_urlType=render&#38;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/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Centerpiece, 12 mm, Epon Charcoal-filled</td>
			<td style="width:64px;">Beckman Coulter</td>
			<td style="width:119px;">306493</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">AN-50 Ti Rotor, Analytical, 8-Place</td>
			<td style="width:64px;">Beckman Coulter</td>
			<td style="width:119px;">363782</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Wizard Plus Miniprep Kit</td>
			<td style="width:64px;">Promega</td>
			<td style="width:119px;">A1470</td>
			<td>used for plasmid purification (Protocol 5.1)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">L-arabinose</td>
			<td style="width:64px;">Gold Biotechnology</td>
			<td style="width:119px;">A-300-500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Glycine</td>
			<td style="width:64px;">DOT Scientific Inc</td>
			<td style="width:119px;">DSG36050-1000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fluorescence Cell cuvette</td>
			<td style="width:64px;">Hellma Analytics</td>
			<td style="width:119px;">119004F-10-40</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Oligonucleotides</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Phusion High-Fidelity DNA polymerase</td>
			<td style="width:64px;">New England Biolabs</td>
			<td style="width:119px;">M0530S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">dNTP set</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:119px;">10297018</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hydrochloric Acid</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">A144-212</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sodium Hydroxide</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">BP359-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amicon Ultra 15 mL 3K NMWL</td>
			<td style="width:64px;">Millipore</td>
			<td style="width:119px;">UFC900324</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Centrifuge Avanti J-26XPI</td>
			<td style="width:64px;">Beckman Coulter</td>
			<td style="width:119px;">393127</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Varian Cary 50 spectrophotometer</td>
			<td style="width:64px;">Agilent Tech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Spectra/Por 1 Dialysis Membrane MWCO: 6 kDa</td>
			<td style="width:64px;">Spectrum Laboratories</td>
			<td style="width:119px;">132650</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amicon Ultra Centrifugal Filter Units 30K</td>
			<td style="width:64px;">Millipore</td>
			<td style="width:119px;">UFC803024</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SDS</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">bp166-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Veriti 96-Well Thermal Cycler</td>
			<td style="width:64px;">Thermo Fisher</td>
			<td style="width:119px;">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

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. ...

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

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 ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...