Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

Small signaling molecules target various effector proteins to control bacterial virulence and human biology. However, these effector proteins are challenging to identify. As a systems biology tool, DRaCALA allows a feasible, rapid, and highly sensitive identification of the unknown effector proteins by using an OVium library.

DRaCALA could be used to study any small signaling molecule as long as it can be labeled with either radioactive isotope or fluorescent dyes. Start by inoculating E.coli into 1.5 milliliters of LB supplemented with 25 micrograms per milliliter chloramphenicol in each well of 96-well deep well plates for an overnight incubation at 30 degrees Celsius and 160 rotations per minute. The next morning, treat the overnight cultures with 500 micromolar IPTG for six hours at 30 degrees Celsius to induce protein expression.

At the end of the incubation, pellet the cells by centrifugation, remove the supernatant, and freeze the samples at minus 80 degrees Celsius. To initiate lysis, resuspend the pellets in 150 microliters of lysis buffer one per well for a 30-minute incubation at minus 80 degrees Celsius before thawing the cells for 20 minutes at 37 degrees Celsius. The lysate should then be stored at minus 80 degrees Celsius before use.

To complete the lysis of cells with overexpressed proteins, resuspend the samples in 40 milliliters of ice cold lysis buffer two and sonicate the samples at a 60%amplitude at two seconds on four seconds off for eight minutes, then clear the lysates by centrifugation. While the samples are being centrifuged, add 500 microliters of homogenized nickel-NTA resin to a standing polypropylene chromatography column. After 15 minutes, wash the settled resin two times with 15 milliliters of ultrapure water and one time with 15 milliliters of lysis buffer two.

At the end of the centrifugation, load the cleared lysate supernatant onto the column. When all the lysate has flowed through the column, wash the column with 30 milliliters of washing buffer. To elute the proteins, add 400 microliter volume of elution buffer to the column and repeat elution three times.

Another 300 microliter volume of the elution buffer to repeat elution three times and combine the eluted proteins in a final volume of 700 microliters. For gel filtration of the eluted sample, after washing a size exclusion column with 25 milliliters of freshly prepared gel filtration buffer, load the entire volume of eluted protein onto the column by using a 500 microliter loop. Run at 500 microliters per minute and collect two to three 500 microliter volume fractions of the respective proteins.

Then use individual spin columns to concentrate each protein and use the Bradford assay kit to measure the protein concentrations according to standard protocols. To synthesize the phosphorus 32 labeled guanosine pentaphosphate, assemble a small-scale Relseq reaction in a screw capped tube as outlined in the table and incubate the reaction in a ThermoMixer for one hour at 37 degrees Celsius and five minutes at 95 degrees Celsius, followed by five minutes on ice. At the end of the incubations, spin down the precipitated protein by centrifugation and transfer the synthesized phosphorus 32 labeled guanosine pentaphosphate containing supernatant to a new screw capped tube.

For phosphorus 32 guanosine tetraphosphate synthesis, add one micromolar GppA to half of the phosphorus 32 labeled guanosine pentaphosphate product in a new screw capped tube and incubate the reaction for 10 minutes at 37 degrees Celsius, five minutes at 95 degrees Celsius, and five minutes on ice. At the end of the incubation, spin down the precipitate by centrifugation and transfer the phosphorus 32 labeled guanosine tetraphosphate containing supernatant to a new tube. To analyze the isolated target proteins, run one microliter of each sample on a thin layer chromatography plate using 1.5 molar monopotassium phosphate as the mobile phase.

After the analysis, place the dried plate in a transparent plastic folder and expose the plate to a storage phosphor screen for five minutes before visualizing and quantifying the data on a phosphor imager. For DRaCALA screening of the target proteins, add 20 microliters of the thawed wholesale lysates to individual wells of a 95-well V-bottom microtiter plate and add 2.5 units of Serratia marcescens endonuclease to each well. After 15 minutes at 37 degrees Celsius, place the lysates on ice for 20 minutes.

Next, mix equal volumes of phosphorus 32 labeled guanosine pentaphosphate and guanosine tetraphosphate and add 1X lysis buffer one to the mixture to obtain a four nanomolar guanosine pentaphosphate solution. Using a multi-channel pipette and filtered pipette tips, mix 10 microliters of the guanosine pentaphosphate mixture with the cell lysate for a five-minute incubation at room temperature. At the end of the incubation, wash a 96X pin tool three times in a 0.01%solution of non-ionic detergent for 30 seconds, followed by 30 seconds of drying on a paper towel per wash before placing the pin tool in the 96-well sample plate.

After 30 seconds, lift the pin tool straight up and place it straight down on a nitrocellulose membrane for 30 seconds. After five minutes of drying, place the nitrocellulose membrane in a transparent plastic folder for storage phosphor screen exposure and visualization by phosphor imaging as demonstrated. To quantify and identify potential target proteins, in the analysis software associated with the phosphor imager, open the gel file of the visualized plates.

To define the spots to be analyzed, use the array analysis function to set up a 12 column by eight row grid. To circumscribe the outer edge of the hole spots, define big circles, export the volume plus background and area of the defined big circles to a spreadsheet. To circumscribe the small inner dots, size down the defined circles, export the volume plus background and area of the defined small circles and save all the data in the spreadsheet.

Position circles to overlap with spots as necessary, resizing the slightly bigger than actual spots. Use the equation to calculate the binding fractions in the spreadsheet and plot the data. Then identify the potential binding proteins in the wells that show high binding fractions compared to the majority of other wells.

In this representative analysis, a plate with relatively low background binding signals in most wells can be observed. The positive binding signal in well H3 exhibited a binding fraction that was much higher than that observed for the other wells due to overexpression of the guanosine pentaphosphate binding protein Hpt. In the representative plates, several wells showed a relatively higher background binding signals as indicated by the relatively strong inner dots observed in many wells, as well as the consistently high binding fractions observed after quantification.

Notably, some targets also gave variable binding fractions, such as the false positives. To clarify further, researchers used purified proteins to test the binding again. Once the target proteins are identified, one can purify them to homogeneity and confirm interaction strength and specificity by using either DRaCALA, isothermal titration calorimetry, or other methods.

Identifying the target proteins of small signaling molecules paves the way to an in-depth understanding of the roles emerging from bacterial virulence to human biology.