The overall goal of these in vivo and in vitro chaperone assays is to characterize the pH-dependent activity of the Escherichia coli chaperone HdeB under acidic pH conditions. Acid activated chaperones have only one job to do, they need to protect proteins against acid induced protein unfolding and degradation. This function is particularly important for enteric bacteria, which have to survive the acidic environment of the stomach if they want to successfully colonize the gut.
The methods that we are presenting here allow you to identify and characterize acid activated chaperones, and define the pH conditions that lead to their activation. After watching this video, you should have a good understanding of how to characterize the chaperones, such as HdeB in vitro and in vivo, which helps you to decipher the mechanism of its activation. These methods have been successfully applied for other acid protective chaperones, such as HDA, and can be further modified to work with other chaperones, and/or stress conditions.
The influence of purified HdeB on the aggregation of thermally unfolding porcine mitochondrial malate dehydrogenase at different pH values is monitored. Dialyzed MDH at four degrees Celsius over night against four liters of buffer C.Then concentrate the protein to approximately 100 micromolar using centrifugal filter units with a molecular weight cutoff of 30 kilodaltons. To remove aggregates, centrifuge the protein for 20 minutes at 20, 000 times G at four degrees Celsius.
Determine MDH concentration by absorbance at 280 nanomolar. Then prepare 50 micro liter aliquots of MDH and flash-freeze the aliquots for storage. Place a one milliliters quartz cuvette into a florescent spectrophotometer equipped with temperature controlled sample holders and stirrers.
Set the excitation and admission wavelength to 350 nanometers. Add the appropriate volumes of pre-warmed buffer D at the desired pH values to the cuvette. The total volume is 1, 000 microliters.
Set the temperature in the cuvette holder to 43 degrees Celsius. Add 12.5 micromolar HdeB to the buffer, followed by the addition of 0.5 micromolar MDH. Begin monitoring light scattering.
Incubate the reaction for 360 seconds to allow sufficient unfolding of MDH. Raise the pH to seven by adding a 0.16 to 0.34 volume of two molar un-buffered dipotassium phosphate, and continue recording light scattering for another 440 seconds. Analyze the data as described in the text protocol.
Transform the plasmid expressing HdeB or the MD vector control, PBAD 18, into strain BB7224 Delta RPOH using chemically competent cells. This strain is temperature sensitive and has to be cultivated at 30 degrees Celsius. After 45 seconds of heat shock at 42 degrees Celsius and prior to plating, incubate the cells at 30 degrees Celsius and 200 RPM.
Perform single colony streak outs of positive clones, and incubate over night at 30 degrees Celsius. Prepare an over night culture in 50 milliliters of LB with Ampicillin, and cultivate the cells at 200 RPM and 30 degrees Celsius. Dilute over night cultures 40 fold into 25 milliliters of LB with Ampicillin.
Grow the bacteria in the presence of 0.5%arabinose at 30 degrees Celsius and 200 RPM to an optical density at 600 nanometers, or OD 600 equal to 1.0, to induce HDEB protein expression. For the pH shift experiments, use LB with Ampicillin and arabinose to dilute the cells to an OD 600 of 0.5. Adjust to the respective pH values by adding appropriate volumes of five molar hydrochloric acid.
After the indicated time points, neutralize the cultures by addition of the appropriate volumes of five molar sodium hydroxide. Monitor the growth of the neutralized cultures and liquid culture for 12 hours at 30 degrees Celsius using OD measurements. To investigate the pH optimum of HdeB's chaperone activity in vitro, native MDH was diluted into pre-warmed buffer of the indicated pH in the presence, or absence, or HdeB.
Following 360 seconds of incubation, aggregation of MDH at 43 degrees Celsius was triggered through neutralization. In the presence of HdeB, the light scattering signal is significantly decreased upon neutralization from pH four, indicating that HdeB prevents the aggregation of MDH. Aggregation measurements are very sensitive to changes in temperature or buffer content.
To eliminate false positive results, the influence of chaperone storage buffer on client aggregation was investigated. The HdeB storage buffer had no effect on MDH aggregation. 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 oxidated stress. Over expression of the 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, PBAD 18. In contrast, clear differences were found in their ability to resume growth upon pH three or pH four treatments, with the HdeB over expressing strain showing reproducibly improved recovery from low pH treatment as compared to the control strain.
Once mastered, the purification can be done in three days, the in vitro scattering assay in one day, and the in vivo survival assays in two days if it is performed properly. When you're doing these experiments, make sure that you consider your experimental design carefully, both in regards to the type of client proteins, their concentrations, and the buffer conditions that you use. The main advantage of this technique is that it helps to gain a more detailed understanding about the mechanism of chaperone activation and its effects in an in vivo context.
And don't forget that studying the pH optimum of an acid protective chaperone can quite challenging. This is because the aggregation behavior of the client protein that you use will likely also change with the pH. So therefore, make sure to choose buffer systems that are suitable for both the chaperone and the client protein.
Following this procedure, other methods like spin down assays by SCS page, can be performed in auto quantified chaperone mediated suppression of client aggregation.
