Single-molecule force spectroscopy can provide enormous insights into molecular biology on the atomic scale by allowing us to manipulate single molecules with force. Here, we demonstrate a constant velocity measurement using atomic force microscope. We can ultimately use the data to determine the protein stability, unfolding rate, and unfolding pathway.
To begin the procedure, attach a non-conductive adhesive tab to a clean, 15 millimeter-wide iron disk. Remove the cover sheet and firmly press a clean, uncoated, or gold-coated glass slide onto the exposed adhesive. Store the slide in a clean, covered Petri dish.
Next, concentrate the purified polyprotein in a centrifugal filter, then reverse the column and elute the protein into the buffer to be used in the experiment. Determine the appropriate protein concentration based on the absorbance at 280 nanometers, and then prepare 100 microliters of a 100 microgram per milliliter solution of the protein. Apply a 60 microliter drop of the protein solution to the center of the slide.
Take care not to let the liquid slip between the slide and the iron disk, as this will lead to uncontrolled sample movements during the experiment. Let the sample sit at room temperature for at least 10 minutes. First, carefully pick up the cantilever by the end and place it in the probe holding cell.
Ensure that the cantilever is firmly seated. Then place the holding cell in the AFM head. Set the head on an inverted microscope stage and connect a battery pack to the AFM head to power the laser.
Set up a camera for the microscope detector to visualize the laser light on a monitor or TV screen. Guided by the microscope, position the laser so that it is directed onto the cantilever tip. Once the sample is ready, flush 10 microliters of the experiment buffer into each port of the probe holding cell.
Decant approximately 40 microliters of fluid from the sample slide and add 40 microliters of buffer. In the meantime, begin preparing the atomic force microscope for the measurement. First, place the slide on the magnet above the piezo motor.
Confirm that the AFM stage is in the elevated position, then place the AFM head on the stage with the cantilever above the sample droplet. Next, place a small piece of paper in front of the AFM photodiode. Adjust the laser until the spot on the paper is bright and focused, and then remove the paper.
Start the AFM software to view the signal from the photodiode. Adjust the AFM head mirror so that the laser signal is maximized in all four photodiode quadrants, and a different signal between the top and bottom quadrant pairs is zero. To begin calibrating the AFM, turn the filter setting for the AFM head signal to full bandwidth, and ensure that the piezo is off.
In the AFM software, measure the average of 512 calculations of the power spectrum, with 1, 024 data points per calculation. Integrate the power spectral density across the first peak, which corresponds to the main mode of vibration for the cantilever. Next, turn on the piezo controller and set the filter to 500 Hertz.
Swiftly move the AFM head down a few hundred micrometers while monitoring the different signal. Continue moving the head down a few hundred micrometers at a time while watching for consistent jumps in the different signal. When the signal jumps start increasing in height, the tip is very close to the sample surface.
Once the deflection signal saturates upon tip contact with the surface, raise the head slightly, then adjust the piezo voltage to move the AFM head up or down by 100 to 5, 000 nanometers to find the sample surface. After that, conduct a pulling experiment with a scan size of about 500 nanometers so that the tip contacts the surface, or about 100 nanometers of piezo motor travel. Use the results to calculate the spring constant and sensitivity.
In the software, set the scan size to 40%larger than the total theoretical size of the unfolding polyprotein. Position the cantilever so that it is 80%of the scan size away from the surface. Set the scan speed to 300 nanometers per second.
Perform a pulling experiment and measure the resulting piezo position and the photodiode signal. Add experiment buffer to the fluid cell hourly throughout the measurement to keep the sample from drying out. The unfolding events in recordings of an I91 polyprotein and of a polyprotein in which three I91 domains flanked the NI10C molecule on each side, were fitted with a worm-like chain model.
Both recordings show the characteristic force and contour length increments of I91, indicating that the AFM calibration was successful. The recordings of the polyprotein containing the N110C molecule showed enough I91 events to confirm that the new events were from the protein of interest unfolding. The mean contour length for the ankyrin repeat was about 10.5 nanometers, and the unfolding forces were between eight and 25 piconewtons.
It's essential to use a polyprotein because the unfolding events of the flanking proteins provide a positive control to fingerprint the single molecule event. Without this, the data is liable to misinterpretation.
