The workflow presented here can help extend our understanding of the cellular role of chaperones during oxidative stress conditions. Specifically, it can help to understand how do chaperones prevent protein aggregation during unfolding conditions. For these two backs of different techniques provide insight into the activity of flag segregated chaperones.
It can also be used to study other protein for interaction such as enzyme social interactions and protein dynamics. Visual demonstration of this method is critical as it involves the usage of different instruments which may be challenging to new users. First, spin down a previously thawed protein sample to remove aggregates.
Then add five millimolar DTT and 20 micromolar zinc chloride and incubate the sample for at least 1.5 hours at 37 degrees Celsius. To remove the DTT equilibrate a desalting column with at 40 millimolar potassium phosphate buffer by filling the column completely with the buffer and letting it drip out. Using tweezers, gently push down on the white disc filter in the column and remove it.
Refill the column with potassium phosphate buffer and centrifuge at 1000 times G for three minutes. Following centrifugation transfer the column into a clean tube and add the protein sample slowly to the middle of the column. Centrifuge the column at 1000 times G for two minutes so that the DTT-free protein is in the flow-through.
Now check the protein concentration and measure its absorbance at 280 nanometers. Distribute half of the protein samples into aliquots. Incubate the aliquots under anaerobic conditions for 20 minutes for complete removal of oxygen.
Then seal the tubes with plastic film and store the samples under cold conditions. Next, add freshly-prepared five millimolar hydrogen peroxide to the remaining protein sample and incubate for three hours at 40 degrees Celsius while shaking. Following centrifugation check the protein concentration then divide the oxidized proteins into aliquots and store them under cold conditions.
Prepare the denatured substrate by incubating 12 micromolar citrate synthase overnight in 40 millimolar HEPES and 4.5 molar guanidinium chloride. On the following day, open the fluorospectrometer software and go to time course measurement. Set the excitation bandwidth to 2.5 nanometers.
The excitation wavelength to 360 nanometers. The emission wavelength to 360 nanometers. The emission bandwidth to five nanometers.
And the data interval to 0.5 seconds. Set the temperature to 25 degrees Celsius. Prepare the sample by adding 1600 microliters of 40 millimolar HEPES to a quartz cuvette.
Insert the cuvette into the sample holder of a fluorospectrometer allowing the sample to reach the desired temperature. After setting the stirring to 600 rpm begin the measurement until the baseline is established. To measure the citrate synthase aggregation in the absence of a chaperone, add 10 microliters of denatured citrate synthetase, 120 seconds into the measurement.
Then continue the measurement for 1200 seconds. To measure the citrate synthase aggregation in the presence of Hsp33 add Hsp33 60 seconds into the measurement. After an additional 60 seconds add 10 microliters of denatured citrate synthetase and continue the measurement for 1200 seconds.
Next, upload the data file to perform data analysis and noise removal by Kfits. To remove any noise choose analysis parameters use automatic best model and select noise is always above signal. Manually remove obvious outliers using the green and red adjustable lines which will filter the noises above the green line and below the red line.
Following this, set the baseline and the fit curve. After applying the noise threshold, download the processed data. Prepare protein substrate complex samples by adding citrate synthetase to Hsp33 at a ratio of 1 to 1.5 at 43 degrees Celsius.
Use at least four steps and incubate the samples for 15 minutes at 43 degrees Celsius after every addition to allow complex formation. Centrifuge the samples at 16000 times G at four degrees Celsius for 30 minutes to remove any aggregates. Then place the samples in 150 microliter glass inserts and transfer them into vials.
Then place them in the proper trays. Hold buffers H and D at 25 degrees Celsius and hold the samples and quenching buffer at zero to two degrees Celsius. Next, manually turn on all cooling units of the mass spectrometer.
Once all cooling systems reach their target temperatures manually switch on both the HPLC and loading pumps. Open the software which controls both pumps as well as the software which controls the mass spectrometer and make sure that the MS is on standby. Following this, disconnect the HPLC outlet valve from the MS source.
Wash the system first with a triple cleaner solution. Then with buffer B and finally with buffer A.Then insert the pepsin column into the system. After washing the system with a steady flow and steady pressure, insert the HPLC outlet valve into the MS source and turn the MS on.
Now open the software. Build a running sequence and enter all time points, sample names and locations in the trays as well as the buffer names and locations. After the run is complete analyze the peptide coverage of the sample by running the non-deuterated control samples through the software.
Transfer data into the HDX Workbench software and analyze the results using the software. Save the selected peptides and continue further for analysis of the deuterium uptake. Chemical denaturation leads to a rapid citrate synthetase aggregation induced by the re-folding of a fully denaturized protein in unfavorable buffer conditions.
On the other hand, the thermal-induced aggregation results from a relatively slow unfolding of the natively folded substrate. In both forms of denaturation, the addition of oxidized Hsp33 completely abolished aggregation, lowering the 360 nanometer readings to negligible values. The presence of a reduced Hsp33 had no effect on the citrate synthetase stability and gave an aggregation curve similar to the one detected in the absence of a chaperone.
The deuteration pattern between the bound and unbound chaperone Hsp33 to its substrate citrate synthetase reveals a client interaction site within the C-terminal redox switch domain of the chaperone. Reduced Hsp33 has a completely ordered structure but becomes functional when it senses oxidative stress. While the reduced bound and unbound Hsp33 produces similar peptide curves the oxidized Hsp33 shows a 30%deuteration difference between the bound and unbound chaperone with specific areas of the bound protein indicating an allosteric hindrance.
Once established the scattering assay can be completed within a few hours while the DHEX methodology can be completed in two to four days if performed properly. While attempting this procedure it is important to plan ahead. Remember that the condition described such as temperature, incubation times and buffer solutions are protein-specific and must be optimized for each protein.
Following this procedure other methodologies such as cross-linking, or precipitation can be performed in order to complement studies of chaperone substrates interactions. In conclusion, this technique has paved the way for researchers in the field of structural biology to explore protein plasticity in molecular chaperones.
