This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.
Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many 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'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's chaperone function 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'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'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'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 ΔrpoH 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.
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.
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.
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.
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.
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.
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's chaperone activity in vitro, native MDH was diluted into pre-warmed (43 °C) buffer of the indicated pH in the presence or absence of HdeB. After 360 sec of incubation, the incubation reaction was neutralized. This neutralization triggers aggregation of MDH at 43 °C 9. Representative results show that the light-scattering signal of MDH in the absence of HdeB increases dramatically upon neutralization due to aggregation of MDH (Figure 1, black line at each pH). In the presence of HdeB, the light-scattering signal is significantly decreased upon neutralization from pH 4 or pH 5 indicating that HdeB prevents the aggregation of MDH (Figure 1, pH 4 and pH 5). In contrast, however, upon neutralization from pH 2 and pH 3, MDH rapidly aggregates to the same extent independent of the presence or absence of HdeB (Figure 1, pH 2 and pH 3), indicating that the pH optimum for HdeB's chaperone activity is between pH 4 and 5. The HdeB storage buffer had no effect on MDH aggregation (Figure 1, buffer control), indicating that the reduced light-scattering signal of MDH in the presence of HdeB at pH 4 is due to its chaperone function. HdeA is chaperone active in its monomeric and unfolded form at pH 2-3 but shows no activity at or above pH 4 10. These results suggest that HdeB has its optimal chaperone activity around pH 4 10.
Figure 1: Chaperone activity of HdeB at acidic pH. 0.5 µM MDH was incubated in pre-warmed buffer D at the indicated pH in the absence or presence of 12.5 µM HdeB for 360 sec at 43 °C. The pH of the samples was then raised to pH 7 (as indicated by the asterisk) by addition of 0.16-0.34 volume 2 M unbuffered K2HPO4, MDH aggregation was measured for an additional 440 sec by monitoring light scattering at 350 nm at neutral pH (blue background). Figure is modified from Dahl et al. 10.This research was originally published in the Journal of Biological Chemistry. Dahl JU, Koldewey P, Salmon L, Horowitz S, Bardwell JC, Jakob U. HdeB functions as an acid-protective chaperone in bacteria. J Biol Chem. 2015, 290(1):65-75. doi: 10.1074/jbc.M114.612986. Copyright the American Society for Biochemistry and Molecular Biology. Please click here to view a larger version of this figure.
To address whether HdeB forms stable complexes also with other client proteins, 3 µM LDH was thermally unfolded at 41 °C in the presence or absence of 30 µM HdeB at different pH conditions. Analysis of the sedimentation behavior of these samples by analytical ultracentrifugation was conducted at 22 °C. When LDH and HdeB were incubated together at pH 7 and 41 °C, LDH remained exclusively tetrameric (molecular weight of 120 kDa) and HdeB remained dimeric. These results indicated that the proteins do not form stable complexes at pH 7, and LDH does not undergo any irreversible changes in oligomerization upon a 20 min incubation at 41 °C (Figure 2, green line). Similarly, HdeB remained dimeric upon incubation at 41 °C and pH 4.0, indicating that low pH incubation of HdeB does not affect its oligomerization state even under heat shock conditions (Figure 2, red line). LDH when incubated at pH 4 and 41 °C in the absence of HdeB rapidly aggregated as indicated by the fact that 40% of LDH sedimented before the first scan was recorded (Figure 2, stated in the upper right corner). The remaining LDH appeared to sediment predominantly as monomer (Figure 2, blue line). In contrast, incubation of LDH and HdeB at pH 4 and 41 °C caused a large proportion of the two proteins to co-sediment (HdeB-LDHC), forming a new species with a molecular weight of 134 kDa. This species likely represents a complex between HdeB dimers and thermally unfolding LDH. Moreover, in the presence of HdeB, no significant LDH aggregation prior to the sedimentation was observed, which is consistent with our in vitro aggregation measurements. These results show that HdeB exhibits chaperone activity in its dimeric form at pH 4.0. This is in stark contrast to HdeA, which is chaperone active in its monomeric form. The very dynamic nature of HdeB that allows it to undergo structural rearrangements between pH 4 and pH 7 is likely sufficient for the activation of HdeB's chaperone function 10.
Figure 2: Detection of complex formation between HdeB and unfolded LDH at pH 4 by analytical ultracentrifugation. 3 µM LDH was incubated in the presence of a 10-molar excess HdeB in buffer D (150 mM KHPO4, 150 mM NaCl) for 15 min at 41 °C at either pH 7 (green line) or pH 4 (black line). For comparison, LDH alone (blue line) or HdeB alone (red line) were incubated for 15 min at 41 °C at pH 4. Analytical ultracentrifugation sedimentation velocity was used to determine the stoichiometry of HdeB, LDH, and the complex formed between HdeB and LDH at different pH conditions. Note that ~40% LDH aggregated prior to the first scan when incubated at pH 4 and in the absence of HdeB (as noted in the upper right corner). Shown is a sedimentation coefficient distribution plot (c(s)) analyzed using the program SEDFIT. Letters indicate the respective oligomeric state of LDH or HdeB, respectively: HdeBD, HdeB dimer; LDHM, LDH monomer, LDHT, LDH tetramer; HdeB-LDHC, HdeB-LDH complex. Figure is modified from Dahl et al. 10. This research was originally published in the Journal of Biological Chemistry. Dahl JU, Koldewey P, Salmon L, Horowitz S, Bardwell JC, Jakob U. HdeB functions as an acid-protective chaperone in bacteria. J Biol Chem. 2015, 290(1):65-75. doi: 10.1074/jbc.M114.612986. Copyright the American Society for Biochemistry and Molecular Biology. Please click here to view a larger version of this figure.
To test whether HdeB supports the refolding of client proteins upon neutralization, HdeB's influence on the refolding of pH-denatured MDH was analyzed. Thermally denatured MDH was incubated at different pH values in the presence or absence of HdeB. After low pH incubation, the pH was neutralized (which initiates MDH refolding), and MDH activity was determined after 2 hr. As shown in Figure 3, significant reactivation of MDH was achieved upon neutralization from pH 4 in the presence of HdeB. No MDH activity was determined when HdeB was absent from the low pH incubation.
Figure 3: HdeB facilitates the refolding of acid denatured MDH to an enzymatically active state. 1 µM MDH was incubated in buffer D at the indicated pH for 1 hr at 37 °C in the absence or presence of 25 µM HdeB. Then, the temperature was shifted to 20 °C for 10 min before the samples were neutralized to pH 7 by the addition of 0.5 M Na2HPO4. Aliquots were taken after 2 hr of incubation at 20 °C and assayed for MDH activity. MDH activity upon neutralization in the absence (white bars) or presence of HdeB (black bars) is shown. Standard deviation derived from at least 3 independent measurements is shown. Figure is modified from Dahl et al. 10. This research was originally published in the Journal of Biological Chemistry. Dahl JU, Koldewey P, Salmon L, Horowitz S, Bardwell JC, Jakob U. HdeB functions as an acid-protective chaperone in bacteria. J Biol Chem. 2015, 290(1):65-75. doi: 10.1074/jbc.M114.612986. Copyright the American Society for Biochemistry and Molecular Biology. Please click here to view a larger version of this figure.
Our in vitro data revealed that HdeB binds different model client proteins at pH 4, prevents their aggregation and facilitates client refolding once neutral pH conditions are restored. To investigate the effects of HdeB in vivo, pH-dependent survival assays were conducted using the temperature-sensitive rpoH deletion strain. This strain lacks most chaperones and is therefore more susceptible to elevated temperature, low pH or oxidative stress 15. Overexpression of HdeB under neutral pH conditions showed no effect on the growth rate of the strain, which grew comparably well to the control strain that harbors the empty vector pBAD18 (Figure 4, untreated). In contrast, we found clear differences in their ability to resume growth upon pH 3 or pH 4 treatment with the HdeB overexpressing strain showing reproducibly improved recovery from low pH treatment than the control strain. In contrast to our in vitro data, however, presence of HdeB had also a significantly protective effect at pH 3. This might be due to the high concentration of HdeB in the cell, which might shift the oligomerization state of HdeB towards dimers even at pH 3. Note that shifting cells to pH 2 or pH 3 resulted in very fast and highly toxic effects, while no significant killing was observed when cells where incubated at pH 4 9,10,22.
Figure 4: HdeB protects E. coli against acidic pH. HdeB (red circles) was overexpressed in BB7224 (ΔrpoH) in the presence of 0.5% arabinose at 30 °C. BB7224 cells harboring the empty vector pBAD18 were used as control (black circles). Upper left panel shows growth of both strains at 30 °C, pH 7. Cells were shifted to the indicated pH by adding 5 M HCl, and incubated for 1 min at pH 2 (upper right panel), 2.5 min at pH 3 (lower left panel) or 30 min at pH 4 (lower right panel). Subsequently, cultures were neutralized by adding appropriate volumes of 5 M NaOH and growth was monitored in liquid media at 30 °C. Figure is modified from Dahl et al. 10. This research was originally published in the Journal of Biological Chemistry. Dahl JU, Koldewey P, Salmon L, Horowitz S, Bardwell JC, Jakob U. HdeB functions as an acid-protective chaperone in bacteria. J Biol Chem. 2015, 290(1):65-75. doi: 10.1074/jbc.M114.612986. Copyright the American Society for Biochemistry and Molecular Biology. Please click here to view a larger version of this figure.
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 overexpression study of HdeB under acid stress conditions, where the rpoH-deficient strain was utilized. This strain lacks most of the chaperones and thus is more sensitive towards various stressors, including elevated temperature, low pH and oxidative stress 15. As alternative, survival studies similar to the ones presented here can be performed in mutant strains that lack the gene of interest.
The experimental design for any chaperone activity or refolding assay has to be carefully considered, both in regards to the type of client, the concentration of the client protein and the buffer conditions. In typical chaperone assays, model client proteins, such as citrate synthase, luciferase, or malate dehydrogenase are denatured in high concentrations of urea or guanidine-HCl, and diluted into denaturant-free buffer to induce aggregation 23,13. Measurements in the presence of chaperones reveal the extent to which they prevent protein aggregation. Alternatively, the clients are thermally unfolded and protein aggregation is monitored. In both cases, light scattering measurements are used as readout for protein aggregation. Active chaperones prevent the aggregation of unfolding client proteins, thereby causing a decrease in the light scattering signal 6. Both of these experimental setups can be combined with low pH incubation. In addition to assessing the molecular chaperone activity by monitoring their ability to prevent aggregation of particular client proteins, the influence of chaperones on client refolding upon return to non-stress conditions can be tested 24. This is especially straightforward when the client proteins possess enzymatic activity, which can be used as quantitative readout for their inactivation and reactivation. While chaperone-mediated refolding is naturally an ATP-dependent process, periplasmic chaperones such as HdeA, HdeB and Spy have been shown to facilitate client refolding in an ATP-independent fashion, consistent with the lack of energy in the periplasm 9,25.
Studying the pH optimum of an acid-protective chaperone such as HdeB is challenging due to various reasons: (i) aggregation behaviors of even well-established chaperone-client proteins such as citrate synthase differ at acidic pH; and (ii) only few buffer systems work in the pH range between 2-5 and are suitable for both the chaperone of interest and the client protein. We decided to use phosphate buffer, although we are aware that this is a non-ideal buffer system under acidic pH conditions. However, phosphate buffer was found to be well-suited to characterize HdeA as acid activated chaperone 9,22. Aggregation measurements are very sensitive towards changes in temperature or buffer content. To eliminate false-positive results, we therefore recommend to always test the influence of chaperone storage buffer on client aggregation (Figure 1, buffer control). Sometimes aggregation of the client protein occurs so fast that even the best chaperone might not be capable of competing with the aggregation process. It is therefore essential to conduct preliminary tests to find the optimal assay conditions. A good example for such a situation is given in our ultracentrifugation experiments where incubation of LDH at temperatures of >42 °C is so fast that even the presence of an excess of HdeB does not prevent LDH aggregation. In addition, the chaperone/co-chaperone ratio or chaperone/client ratio has to be determined carefully 23. We started using a fairly high HdeB:MDH ratio of 50:1 in preliminary experiments and that helped us in identifying pH 4 as the optimal pH for the chaperone activity of HdeB. We then continued analyzing HdeB:MDH ratios between 1:1 and 50:1 at pH 4, identifying 25:1 to be the most effective ratio. In contrast, HdeA suppressed MDH aggregation as 10:1 chaperone:client ratios 6,9,10,22. Thus, we conclude that HdeA, in comparison to HdeB, is more effective in suppressing MDH aggregation as lower chaperone:client ratios were sufficient to completely suppress MDH aggregation. Another approach to investigate chaperone-mediated suppression of protein aggregation involve spin-down assays, in which client aggregates are removed by centrifugation and quantified by SDS PAGE. This approach is also suited for monitoring the influence of chaperones on protein aggregation in vivo. Mutant strains that either overexpress or lack the chaperone of interest are exposed to protein unfolding stress conditions. Subsequently, the cells are lysed and soluble and aggregated fractions are separated and quantified 15,16,26.
For detection of the client-chaperone complex, we applied analytical ultracentrifugation. It shall be noted here that based on the experimental setup it is not possible to directly quantify the amount of HdeB and LDH monomers bound in this complex, as both proteins absorb at 280 nm. If desired, the stoichiometry of the chaperone-client complex can be determined by separately labeling chaperone and client protein with a chromophore, whose excitation maximum lies within the visible range. Alternatively, the stoichiometry of clients to chaperones within complexes can be determined by using native PAGE coupled with quantitative western blot.
By following the protocols presented here, we were able to characterize two molecular chaperones, HdeA, and HdeB 9,10,22. In general, these assays can be also used to investigate the role of potential inhibitors of molecular chaperones in protein refolding in vitro and in vivo or can be applied to test synthetic chaperones for their ability to prevent client aggregation under acid stress. In addition, the protocols presented here can be used for analyses of point-mutations and/or truncated variants of the acid-activated chaperones in order to shed light into the mechanism of their activation.
The authors have nothing to disclose.
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).
NEB10-beta E. coli cells | New England Biolabs | C3019I | |
Ampicillin | Gold Biotechnology | A-301-3 | |
LB Broth mix, Lennox | LAB Express | 3003 | |
IPTG | Gold Biotechnology | I2481C50 | |
Sodium chloride | Fisher Scientific | S271-10 | |
Tris | Amresco | 0826-5kg | |
EDTA | Fisher Scientific | BP120-500 | |
Polymyxin B sulfate | ICN Biomedicals Inc. | 100565 | |
0.2 UM pore sterile Syringe Filter | Corning | 431218 | |
HiTrap Q HP (CV 5 ml) | GE Healthcare Life Sciences | 17-1153-01 | |
Mini-Protean TGX, 15% | Bio-Rad | 4561046 | |
Malate dehydrogenase (MDH) | Roche | 10127914001 | |
Potassium phosphate (Monobasic) | Fisher Scientific | BP362-500 | |
Potassium phosphate (Dibasic) | Fisher Scientific | BP363-1 | |
F-4500 fluorescence spectrophotometer | Hitachi | FL25 | |
Oxaloacetate | Sigma | O4126-5G | |
NADH | Sigma | N8129-100MG | |
Sodium phosphate monobasic | Sigma | S9390-2.5KG | |
Sodium phosphate dibasic | Sigma | S397-500 | |
Lactate dehydrogenase (LDH) | Roche | 10127230001 | |
Beckman Proteome Lab XL-I analytical Ultracentrifuge | Beckman Coulter | 392764 | https://www.beckmancoulter.com/wsrportal/wsrportal.portal?_nfpb=true&_windowLabel=UCM_RENDERER&_urlType=render&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/ |
Centerpiece, 12 mm, Epon Charcoal-filled | Beckman Coulter | 306493 | |
AN-50 Ti Rotor, Analytical, 8-Place | Beckman Coulter | 363782 | |
Wizard Plus Miniprep Kit | Promega | A1470 | used for plasmid purification (Protocol 5.1) |
L-arabinose | Gold Biotechnology | A-300-500 | |
Glycine | DOT Scientific Inc | DSG36050-1000 | |
Fluorescence Cell cuvette | Hellma Analytics | 119004F-10-40 | |
Oligonucleotides | Invitrogen | ||
Phusion High-Fidelity DNA polymerase | New England Biolabs | M0530S | |
dNTP set | Invitrogen | 10297018 | |
Hydrochloric Acid | Fisher Scientific | A144-212 | |
Sodium Hydroxide | Fisher Scientific | BP359-500 | |
Amicon Ultra 15 mL 3K NMWL | Millipore | UFC900324 | |
Centrifuge Avanti J-26XPI | Beckman Coulter | 393127 | |
Varian Cary 50 spectrophotometer | Agilent Tech | ||
Spectra/Por 1 Dialysis Membrane MWCO: 6 kDa | Spectrum Laboratories | 132650 | |
Amicon Ultra Centrifugal Filter Units 30K | Millipore | UFC803024 | |
SDS | Fisher Scientific | bp166-500 | |
Veriti 96-Well Thermal Cycler | Thermo Fisher | 4375786 |