This method can help answer key questions about phosphotransfer enzymes including enzyme substrate specificity and reaction kinetics. The main advantage of this technique is that it allows for rapid collection of multiple data sets that are very consistent, enabling statistically robust quantification of enzyme activity. Visual demonstration of this method is critical because spotting the reactions onto the TLC plates and quantifying the radioactive species in the reactions is much easier to demonstrate than to explain.
In addition to Astha, demonstrating the procedure will be Asia Poudel, another graduate student from my laboratory. Begin this protocol with inducible overexpression of a histidine-tagged protein as described in the text protocol. Prepare one milliliter of nickel nitriloacetic acid resin in a gravity column to purify the protein.
The day before use, equilibrate the column overnight at four degrees Celsius with two milliliters of equilibration buffer. The following day bring the column from four degrees Celsius to room temperature prior to loading the clarified lysate and let it stand for approximately two to three hours. Then resuspend the pellet in the lysis buffer.
Sonicate the cells on ice for 10 times 10-second intervals, pausing 30 seconds between pulses. Clarify the lysate by centrifugation at 3, 080 times g for 30 minutes at four degrees Celsius using a microcentrifuge. Prepare the clarified lysate with equal volumes of lysis buffer, then apply the prepped, clarified lysate to the column and collect the flow-through.
Reapply the clarified lysate flow-through to the column and collect the secondary flow-through. Then wash the column with five milliliters of wash buffer one and collect the flow-through. Wash the column again with five milliliters of wash buffer two and collect the flow-through.
Now apply two milliliters of elution buffer. Collect flow-through in two fractions of one milliliter each. During protein purification, inclusion of magnesium chloride in the purification buffers, a modification to the manufacturer's protocol, is essential for enzymatic activity.
To qualitatively assess protein purification by SDS-PAGE, run 20 microliter aliquots of all column fractions on a 4%stacking, 10%running polyacrylamide gel for 60 minutes at 170 volts. Stain the gel with 0.1%Coomassie blue at room temperature for five hours, rocking gently on a benchtop rocker. Then destain the gel in 40%methanol-10%glacial acetic acid overnight at room temperature, rocking on the benchtop rocker.
Dialyze the eluted fraction two against dialysis buffer at a 200:1 ratio using a one-milliliter dialysis device with a 20-kilodalton molecular weight cutoff overnight at four degrees Celsius. Determine the concentration of dialyzed protein sample by measuring absorbance at 280 nanometers and using the calculated molar extinction coefficient. Store 100-microliter aliquots of the dialyzed protein sample at minus 80 degrees Celsius until use.
Prior to performing the reaction, prepare PEI cellulose thin layer chromatography plates by washing them in deionized water. Place the plates in a glass chamber with double-distilled water to a depth of approximately 0.5 centimeters. After allowing the water to migrate to the top of the plate, bring the plates out of the glass chamber and leave on a benchtop rack to dry overnight.
Mark the dried plates two centimeters from one edge with a soft pencil to indicate where the samples will be applied for TLC. For two-microliter samples, apply samples no less than one centimeter apart. When planning experiments, always leave one spot on each plate unused to serve as a blank lane for sample quantification.
To perform the enzyme activity assay, prepare individual reactions using gamma P32 ATP as described in the text protocol. Add the RSH after the other components have been mixed, as the addition of RSH to the nucleotide-containing mix initiates the enzymatic activity assay. To control for ATP hydrolysis from contaminating nuclease activity, assemble a 10-microliter reaction containing no protein and incubate it in parallel.
Spot two-microliter samples at T equal zero and at the end of the experiment to ensure that ATP was not hydrolyzed in the absence of protein. Immediately upon addition of RSH, remove two microliters and spot it onto the labeled PEI-cellulose plate as the T equals zero minute sample. Incubate the reaction at 37 degrees Celsius, removing two-microliter aliquots at desired time points.
After all aliquots have been collected, perform thin layer chromatography by filling the chromatography chamber with 1.5-molar monobasic potassium phosphate to a depth of 0.5 centimeters. Immerse the bottom edge of the plate in solvent and allow the solvent to migrate to the top of the plate over approximately 90 minutes. Remove the plate from the chromatography tank and place it on a benchtop drying rack to air dry overnight.
After the plate is dry, wrap the plate in plastic film to avoid transfer of radioactive material to the imaging cassette and analyze by autoradiography. Expose the PEI-cellulose plate containing separated reactions to a phosphorimager cassette for four hours at room temperature. Following exposure, image the cassette on a phosphorimager.
Using imaging software with a graphical user interface, draw regions of interest or ROIs by first selecting Draw a Rectangle, then use the mouse to draw rectangular ROIs around one entire lane and the ATP and guanosine-tetraphosphate spots contained within that lane. Use the Select, Copy, and Paste commands to draw identical ROIs within the other lanes to ensure that the ROIs are measuring signal within identical areas in each lane. Include ROIs from an unused lane to be used as blanks.
Using the Analyze, Tools, ROI Manager, Add commands of the imaging software, select all of the ROIs drawn on the PEI-cellulose plate. Now use the Analyze, Set Measurements, Measure commands to quantify the signal intensity within each ROI and export the measurements as a spreadsheeet. In the spreadsheet, subtract blank ROI values from experimental signals.
Converting the data to the percent guanosine-tetraphosphate synthetized is critical, because it ensures that data sets collected on different days with different batches of protein or different gamma P32 ATP are consistent and can be pooled for statistical rigor. This cartoon demonstrates how regions of interest are used to define a lane that contains the total radioactive signal in a sample and the component radioactive ATP and guanosine tetraphosphate regions of interest. ATP and guanosine tetraphosphate signals are normalized to the total signal rather than reporting the absolute values because pipetting error or decay of the radioactive substrate can affect the total signal in the sample but do not affect enzyme activity.
Shown here is an actual TLC plate of a reaction carried out using purified C.difficile RSH. A control reaction containing no protein allows quantification of uncatalyzed ATP hydrolysis while a blank lane allows accurate signal quantification. In the ATP and guanosine-tetraphosphate spots, the enzyme transfers the radioactive phosphate from ATP substrate to GDP precursor, creating radioactive guanosine tetraphosphate.
This confirms that the putative guanosine tetraphosphate synthetase from C.difficile is an active enzyme. By sampling the reaction at intervals, the progress can be observed. Here the raw signal is observed at each time point.
ATP is decreasing and guanosine tetraphosphate is increasing. Shown here is the normalized data. The error bars are much smaller because normalization accounts for any pipetting error.
While attempting this procedure, it's important to be careful not to scratch the resin on the TLC plate, as this can interfere with solvent migration. This is a very accessible technique with straightforward data analysis that can quickly provide robust quantification of enzyme activity. We've used it to confirm that Clostridium difficile synthesizes guanosine tetraphosphate, which has never previously been reported.
Don't forget that working with radioactivity can be extremely hazardous and you must follow your institution's rules for the storage and use of radioactive materials while performing this procedure. This method can provide insight into guanosine tetraphosphate synthesis but it can also be applied to other phosphotransfer reactions, including protein kinase activity and cyclic diguanylate synthesis.
