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1. 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.
- To prepare MDH, dialyze MDH at 4 °C overnight against 4 L buffer C (50 mM potassium phosphate, 50 mM sodium chloride (NaCl), pH 7.5) and concentrate the protein to approximately 100 µM 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 an ammonium sulfate solution. - To remove aggregates, centrifuge the protein for 20 min at 20,000 x g at 4 °C. Determine MDH concentration by absorbance at 280 nm (ε280 nm= 7,950 M-1 cm-1). Prepare 50 µl aliquots of MDH and flash-freeze the aliquots for storage.
- Place a 1 ml quartz cuvette into a fluorescence spectrophotometer equipped with temperature-controlled sample holders and stirrers. Set λex/em to 350 nm.
- Add appropriate volumes of pre-warmed (43 °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 °C. The total volume is 1,000 µl.
- Add 12.5 µM HdeB (or alternatively, the same volume of HdeB storage buffer for the buffer control) to the buffer, followed by the addition of 0.5 µM MDH. Begin monitoring light scattering. Incubate the reaction for 360 sec to allow sufficient unfolding of MDH.
- 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.
- Set the extent of MDH aggregation that is recorded in the absence of the chaperone at a defined time point after neutralization (hereafter 500 sec, when the maximal light scattering of MDH was observed) to 100%. Normalize HdeB's activity to the light scattering signal of MDH in the absence of HdeB at each indicated pH value.