The overall goal of this procedure is to study the effect of mechanical load on the proteolytic cleavage of single proteins in a highly parallel and cost-effective manner. In this demonstration, the cleavage of collagen by matrix metalloproteinase one is examined in the first step, collagen trimmers are attached to the glass cover slip of a flow cell, followed by the attachment of micrometer size magnetic beads to the individual collagen molecules. The second step is to mount the flow cell into the magnetic tweezers apparatus and check that non-specifically attached beads have been removed from the cover slip surface.
Next, the protease is added to flow cells. The final step is to determine the proteolysis rate by monitoring bead detachment in real time. By measuring bead detachment rates at varying protease concentrations and forces, it is possible to derive standard zoological kinetic parameters using minute amounts of substrate and protease.
In addition, this technique provides unique information about the effect of mechanical force on the rate of proteolysis and on the structural changes in the substrate protein that accompany cleavage. The main advantage of this technique compared to other methods like optical T traces and atomic force microscopy, is that this technique is high throughput and cost effective. Though this method can provide insight into the effective force on the proteolytic cleavage of single proteins, the instrumentation and techniques can be applied to a broad range of biophysical problems, including the effect of force on protein folding and confirmation.
Begin flow cell preparation by cleaning cover slips using sonication. Add the cover slips to a small glass container capable of holding them that fits in the ator. Fill the container with isopropanol and sonicate in a bath ator for 20 minutes the the isopropanol and rinse the cover slips with copious quantities of deionized water.
Fill the container with water and sonicate for 20 minutes. After sonication dry the cover slips in a stream of filtered dust-free air. Inspect the cover slips and use only the ones that are free of smudges.
Gently flame the cover slips for a few seconds to clean them of any remaining dust and moisture by passing the cover. Slips through a gas flame produced by a bunsen burner, taking care not to warp the cover slip due to excess heat. This step removes residual surface contaminants.
Next, use double-sided scotch tape to attach the two cover slips together. By first cutting the tape approximately three centimeters in length and 0.3 centimeters in width, attach the tape to a 22 by 40 millimeter cover slip, leaving a 1.6 centimeter channel in the middle. Use a pipette tip to gently press down on the cover slip to ensure that the tape has properly adhered.
The magnetic tweezers apparatus was constructed by this lab using an aluminum L bracket with a one millimeter diameter pinhole and mounted on a vertical Z translator to modulate the height of the tweezers. Two permanent rare earth magnets were attached to the bracket on either side of the pinhole to create the magnetic field gradient. Proper calibration of the magnetic trap is essential to this procedure.
To ensure success, we use two different methods as outlined in this section. To calibrate the magnetic trap For calibration use previously prepared DNA from Lambda phage labeled at its five prime and three prime ends with biotin and doxygen in as described in the written protocol. To attach the DNA to the flow cell first, fill the flow cell with anti-D oxygen and antibody solution and allow the antibody to adhere to the glass surface for 15 minutes.
Next, ate the surface of the flow cell to prevent non-specific sticking of DNA by adding a BSA solution to the flow cell. Note that for this ation step and all subsequent steps, the reagent volume added to the flow cell should be at least twofold. The volume of the flow cell, which in this demonstration is 75 microliters, incubate the flow cell at room temperature for 45 minutes.
After repeating this step, add 50 pico molar of functionalized lambda DNA and allow it to attach to the cover slip for 15 minutes, wash away excess DNA with one XPBS. Finally, add streptavidin coated super paramagnetic beads and allow them to attach to the immobilized DNA for 15 minutes. In this demonstration, 2.8 micrometer beads are used.
Wash away excess beads with one XPBS. Next, perform magnetic tweezers calibration by moving the permanent magnets to a position that is far away from the sample surface. Then place the prepared flow cell in the microscope.
Move the permanent magnets into position above the flow cell. Choose the focal plane of Lambda DNA functionalized beads by focusing on the beads away from both glass surfaces. The correct focal plane is one in which the desired beads have a bright center.
Take a video of bead motion that 80 hertz are greater for 500 frames. Move the magnet closer to the sample surface and record a video of the bead motion at each position. For distances beyond five millimeters, move in one millimeter increments for distances between one and five millimeters.
Move in 0.5 millimeter increments for distances less than one millimeter, move in 0.25 millimeter increments For each dataset, track the center of the beads using A 2D Gaussian fit. Calculating the force via brownian fluctuations as described in the written procedure. Confirm the accuracy of the forces by checking the force calculation via a power spectrum Lian fit.
In order to find the roll off frequency, the relationship between applied force and roll off frequency can be found in the text prior to collagen peptide attachment to flow cells, express and purify the collagen peptide and MMP one proteins. Begin by adding the anti mic antibody solution to the flow cell and incubate at room temperature for 20 minutes to allow the anti mic to attach to the surface of the flow cell ate the flow cell to prevent non-specific attachment of proteins using five milligram per milliliter. BSA Add 150 picomolar collagen peptide and allow it to attach to the antibody via the M ttag for 45 minutes.
Wash away excess collagen using PBS buffer before adding 2.8 micrometer beads to the flow cell. They should be separated from the strep divid and coated bead solution using strong magnets, then resuspended in PBS. This process should be repeated three times.
This step is essential to get the 2.8 micrometer beads to bind to surface immobilized collagen peptide. Next, add strep divid streptavidin coated super paramagnetic beads and allow them to attach to the collagen molecules for 45 minutes. Once the flow cell is assembled with collagen peptide and magnetic beads, image the flow cell in the magnetic trap.
Under low force, a subpopulation of the beads is sometimes weak, adhered to the surface in a non-specific fashion. The application of modest force ensures that these beads are liberated. Add activated enzyme to the flow cell.
In this case, MMP one was pre activated by adding 3.5 millimolar A PMA and incubated at 37 degrees Celsius for three hours. As soon as a flow cell is reintroduced into the magnetic tweezers apparatus, record a video spanning a few fields of view, typically yielding several hundred attached beads. This measurement corresponds to time equals zero.
Repeat the same process of recording several fields of view at regular time points until all the beads detach or no further proteolysis is discernible. Magnetic tweezers were calibrated for one micrometer and 2.8 micrometer beads. Using both the magnitude of the observed brownian fluctuations and calculation of the roll-off frequency at varying magnet positions in the forced proteolysis experiments, the normalized number of beads remaining can be plotted as a function of time to find the proteolysis rate.
And this process can be repeated for varying enzyme concentrations and forces. Proteolysis rates depend on the applied force. The rates of proteolysis were determined at one pico Newton 6.2 Pico Newtons, and 13 Pico newtons.
The fraction of beads unpro lies at greater than 15 minutes, remains approximately constant at 0.25 across different experiments. In our case, the assay allowed us to measure the catalytic efficiency of MMP one as a function of applied force. By analyzing the data, we found that the collagen triple helix must locally unwind prior to proteolysis.
Once mastered, this technique can be performed in five to six hours if done correctly. It is important to remember that at least 30 to 50 beads per fuel of view are required to get good statistical significance of your data. Don't forget that A PMA can be extremely hazardous and precautions such as gloves, lab coats, and face mask should always be used while performing this experiment.
After watching this video, you should have a good understanding of how to measure the effect of force on proteolytic cleavage of single proteins using magnetic tweezers. The magnetic tweezers are a great tool for understanding single molecule biophysical experiments. This technique can be extended to other protease and substrate combinations.
In addition, similar techniques can be used to understand protein structural dynamics as well as understanding other proteins that bind or modify RNA and or DNA.
