The overall goal of the following experiment is to measure kinetics of protein, protein interactions and the allosteric effects of small ligands on those interactions using biolayer interferometry, BLI. This is achieved by preparing one of the proteins with specific biotin and immobilizing it on the surface of strep. Aden coated biosensors in parallel.
As a second step, the sensor bound protein is exposed to buffer containing its binding partner, and binding is measured in real time by its effect on optical interference at the sensor surface. Next, each sensor is transferred to buffer without the binding partner in order to measure realtime dissociation of the protein. Protein complexes.
Results obtained allow determination of the kinetic rate constants for binding and dissociation of the interacting proteins and thus their binding affinity. Further results show that BLI can detect the effects of small ligands on the rate of dissociation of the protein protein complex. The main advantage of this technique over existing methods such as surface plasma resonance, is that BLI is not overly sensitive to refractive index changes.
So the binding and association steps can include changes in small ligands to test their effects on the protein-protein interactions in real time, Though, this method can provide insights into kinetics of protein-protein interaction. It can also be applied to different combinations such as protein binding to immobilized DNA or RNA or liposome binding to an immobilized protein. Begin this experiment by setting up the BLI instrument as described in the text protocol.
Then set up the experimental design in the data acquisition software. Select new kinetics experiment in the experiment wizard tab. This presents a tabbed menu with all steps that must be defined on tab one.
Define the columns to be used. On the 96 well sample plate. Assign columns to contain buffer, the protein to be immobilized or the binding partner.
For each association, well enter the concentration of binding partner to be used. On tab two, define all steps needed for the assay. These include baseline loading association and dissociation of binding partner.
Individual assay steps are linked with columns of wells on the sample plate by selecting the assay step and then double clicking on the respective column. This programs the sensors to be moved from one column of wells to the next. During the assay, begin with a brief baseline step linked to the first column of wells with assay buffer to establish initial BLI signals in the 96 well plate.
Then link the loading step to wells that will contain the biotinylated protein. Use the threshold function to achieve a predetermined level of binding. Set the threshold option so that all sensors will be moved to the next column of wells.
When any one of the sensors reaches the threshold, include another baseline step in buffer to wash away non immobilized biotinylated proteins from the sensors and establish new stable baseline signals for basic assays to determine global binding and dissociation rate constants include an extra baseline step with sensors in a new column of buffer wells before the association. Step next, link the association. Step to the column of wells that contain varying concentrations of the binding partner.
Adjust the step time so that at least a saturating concentration of the binding partner should reach equilibrium binding signal. For the dissociation step, designate the sensors to return to column four, the same buffer wells as used for the extra baseline before association. The times for association and dissociation steps can be adjusted during the experiment if rates are slower or faster than anticipated.
On tab three, indicate locations in the sensor tray that will contain pre wetted sensors for the assay. Tab four allows graphical review of the program. On tab five, enter necessary details including location of data files and the desired temperature.
For the experiment. Pre-wet the streptavidin coated sensors for at least 10 minutes. To remove their protective sucrose coating, remove the sensor containing rack from the sensor tray and insert a 96 well plate in the bottom of the tray sliding one corner of the plate into the orienting notch on the tray.
For the column of sensors to be used, add 200 microliters of assay buffer per well. In that column of the 96 well plate return the rack of sensors to the tray in the correct orientation as assigned During the programming, fill wells of the sample plate with assay buffer or the appropriate protein dilutions. Avoid introducing bubbles as they can cause noise in the optical signal.
Include one or more reference wells that omit either biotinylated protein or binding partner. Open the door of the instrument and note the light output from the sensor arm. Insert the sensor tray onto the stage with the trays tabs inserted into the slots of the stage.
Insert the sample plate into the plate holder. Make sure that the plate is seated flat and in the correct orientation as indicated on the plate holder. Close the door and start the assay from the data acquisition program.
If sensors still require pre wetting, select the option to delay starting the experiment. Finally, once all sample preparation is complete and both the sample plate and sensor tray are loaded in the instrument, click the go button to run the assay. After the assay is run open the data analysis software and load the folder containing the data.
Click the processing tab to see a stepwise processing menu and the raw kinetic data with each sensor assigned a different color under data selection. Click the sensor selection button on the sample plate map. Designate the wells for one or two control sensors as reference wells on the processing menu.
Check the subtraction box and select reference wells to subtract reference signal from every other sensors signal. Align all traces to Y equals zero by using the align y axis step. Then select baseline as the alignment step for time range.
Enter the last 10 seconds of that baseline. Clicking within that baseline step will show the alignment. Next, check the inter step correction box to minimize signal shifts between the association and dissociation steps.
Select a line to baseline or dissociation. Select the KY gole filtering function in most cases, and click process data to proceed visually inspect the final process data in the lower right panel. To analyze the data, click the analysis tab in data analysis for step to analyze, choose association and dissociation.
For model, select one to one for fitting Choose global. For group by select color, select r max unlinked by sensor to allow independent fitting of the maximal signal response upon saturating binding of the partner to the immobilized protein. Finally, click fit curves to start the nonlinear regression analysis.
Examine the fitting results, which include overlay of regression curves with sensor data traces, plots of fitting residuals, and a table with determined parameter values and statistics. Real-time binding and dissociation BLI kinetics are shown here. This experiment was started with a 10 minute baseline step since the sensors had been pre-wet, only briefly, biotinylated epsilon was then loaded on the sensors.
No detectable dissociation of epsilon occurred throughout all remaining steps as seen from the reference curve, which had no binding partner added. A second reference sensor was devoid of a mobilized protein and showed low non-specific binding of the binding partner. Various concentrations of F1 minus epsilon were used in the association step for sensors A through F in order to fit results globally and get the best values for the association rate constant to association rate constant and the equilibrium dissociation constant KD results were processed, yielding the blue data traces.
In this case, the reference sensor trace was subtracted from sample traces to correct for non-specific binding of the binding partner and system signal drift. In the data analysis step, all curves were globally fit with a one to one, one-to-one model. The global fit assumes complete dissociation of the binding partner.
A different experiment is shown here in which the enzyme was bound to sensor immobilized epsilon in the presence of one millimolar, A-T-P-A-D-T-A. This predisposes most F1 epsilon complexes to assume the non inhibitory confirmation, which dissociates readily sensors were then moved to dissociation wells containing assay buffer with different ligands. This demonstrates the effects of different ligands on the confirmation of epsilon.
For example, exposure to magnesium A GP and phosphate dramatically slowed net dissociation indicating that epsilon shifted confirmation to the tightly bound inhibitory state. When first setting up BLI assays, it's important to include controls to test for non-specific binding of protein to the sensors and whether non-specific binding varies significantly with protein concentrations to be used. After watching this video, you should have a good understanding of how to design program and analyze BLI assays for measuring the kinetics of protein protein interactions and appreciate that.
BLI allows a direct test for the effects of small ligands on the protein protein interactions.
