Our research focuses on replicating established model systems, validating them through additional assays and adapting these methodologies to suit our specific research context. We aim to demonstrate a versatile approach that can be applied to various projects. The current experimental challenge lies in strategically selecting the appropriate assay format and conditions based on project specific requirements and existing knowledge gaps.
This is essential for achieving efficient and accurate answers in our experiments. Our protocol provides a streamlined approach for seamless adaption to various PROTACS systems. Furthermore, we conduct a thorough evaluation of each method, highlighting their respective pros and the cons.
We offer informed instructions on choosing the most suitable format based on specific conditions. To begin, prepare solutions for the cell, syringe, and for cleaning. Add protein or buffer samples to three consecutive wells on a 96-well plate.
Now, add 100%DMSO accordingly so that the final concentration is 2%Run all four titrations on the microcalorimeter. Each titration should consist of an initial injection of 0.4 microliters of protein at 2 microliters per second, followed by 19 injections of 2 microliters syringe solution at a rate of 2 microliters per second at 122nd intervals for a total of 20 injections. Execute all experiments at 25 degrees Celsius while stirring at 600 RPM.
For data analysis, fit the data to a single binding site model to obtain the stoichiometry and the dissociation constant and the enthalpy of binding. The characterization of VHL MZ1 binary complex and VHL MZ1 Brd4 ternary complex was performed. Positive cooperativity was observed.
Begin the biolayer interferometry or BLI experiments with streptavidin-coated sensors at room temperature. Ensure the sensors are stationary in a single row. Pipette 200 microliters of the sample into the wells.
Next, immerse the sensor in a solution containing VHL for 80 seconds to load the sensor to 1-3 nanometers. Now, dip into the buffer for 60 seconds to establish the first baseline phase. For the immobilization phase, dip the sensors into the VHL protein for 80 seconds.
Then, dip into the buffer again for 60 seconds to establish the second baseline phase. Next, dip into a fixed concentration of the following injections for 300 seconds. Finally, dip the sensors into the buffer once more for 600 seconds to establish the dissociation phase.
Use the instrument software to analyze the data and estimate the on-rate, off-rate, and equilibrium dissociation constant. The characterization of VHL MZ1 binary complex and VHL MZ1 Brd4 ternary complex was performed. MZ1 was found to mediate the formation of the ternary complex.
To perform SPR for VHL MZ1 interactions, first activate the streptavidin chip as per the manufacturer's instructions. To make the highest concentration, add a DMSO-free buffer to the MZ1 to ensure a 2%DMSO concentration. Now, pipette 50 microliters of the highest concentration solution into a well containing 100 microliters of the running buffer.
Mix thoroughly to set up the second highest concentration. Then transfer 50 microliters of the solution to the next 100 microliters of running buffer. Mix well to prepare the third highest concentration and so on.
Use self-adhesive transparent plastic foils compatible with polypropylene microplates to cover the plate. Initiate the SPR using the multi-cycle setup. Set the mode at high performance, the contact time at 120 seconds, the dissociation time at 300 seconds, and the flow rate at 50 microliters per minute.
Use the evaluation software provided by the instrument manufacturer to execute the data analysis. To perform SPR for VHL MZ1 Brd4 ternary complex, first activate the streptavidin. Inject VHL solution to immobilize it to approximately 100 resonance units.
For the negative control using Brd4 only, prepare the highest concentration of 25 micromolar in the running buffer with a volume of 200 microliters. To the next four wells, add 160 microliters each of 2 micromolar Brd4. Transfer 40 microliters from well A5 to A4.Then pipette the well to mix thoroughly.
Similarly, transfer 40 microliters from well A4 to A3, continuing this process until reaching A1.For ternary complex formation with MZ1 and Brd4, pipette out 196 microliters of 25.5 micromolar Brd4 solution. To this, add 4 microliters of 20 micromolar MZ1 prepared in 100%DMSO solution. Pipette 160 microliters of Brd4 at 2 micromoles in the running buffer to the next four wells on the left.
Next, transfer 40 microliters from well B5 to B4 and pipette to mix thoroughly. Continue to transfer the solution from each well until B1.Now run the SPR to the single cycle setup with a contact time of 100 seconds, a dissociation time of 720 seconds, and a flow rate of 50 microliters per minute. The characterization of VHL MZ1 binary complex and VHL MZ1 Brd4 ternary complex was performed.
Positive cooperativity was observed. Characterization of the cereblon, PROTAC, and PPM1D system was performed by SPR. PPM1D experiments use a nickel NTA chip and the chip surface is regenerated after each compound injection.
BRD-2512, which only binds to cereblon, and BRD-4761, which only binds to PPM1D, show a negligible binding response. BRD-5110 induces the formation of a ternary complex between PPM1D and shows a hook effect at high compound concentrations.
