Assessing Bacterial Chaperone Activity via Thermal Unfolding of a Model Protein

Begin by placing a cuvette into a fluorescence spectrophotometer equipped with a temperature-controlled cuvette holder and stirrer.

Add pre-warmed buffer at a moderately acidic pH and set the holder temperature to simulate stress conditions.

In the test sample, add an acid-protective chaperone derived from E. coli.

In the control sample, add an equal volume of buffer.

Next, introduce a temperature- and acid-sensitive substrate protein into both samples.

Begin monitoring light scattering.

Incubate the samples under stress conditions to induce substrate unfolding.

At the moderately acidic pH, the chaperone becomes active and stabilizes the unfolded protein.

Add a pH-neutralizing salt solution and continue recording light scattering.

In the control sample, the return to neutral pH causes the unfolded protein to aggregate, increasing light scattering.

In the test sample, the chaperone promotes substrate refolding, which causes reduced light scattering compared to the control.

Compare the light scattering signals to assess chaperone activity.

Place a one milliliter quartz cuvette into a fluorescence spectrophotometer equipped with temperature controlled sample holders and stirrers.

Set the excitation and emission 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 unbuffered dipotassium phosphate and continue recording light scattering for another 440 seconds. Analyze the data as described in the text protocol.