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1Department of Chemical Engineering, Stanford University
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In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.
Adhikari, A. S., Chai, J., Dunn, A. R. Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers. J. Vis. Exp. (65), e3520, doi:10.3791/3520 (2012).
The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.
1. Flow Cell Preparation
2. Magnetic Tweezers Setup and Calibration
3. Collagen Peptide Attachment to Flow Cells
In order to provide a concrete example for the application of our methodology, we describe recent work in our laboratory that characterizes the proteolysis of a trimeric collagen model peptide. We anticipate that this general approach may be broadly applied to other proteins and polynucleotides.
4. Force Proteolysis Assay
5. Representative Results
The above protocol describes a novel use of magnetic tweezers (Figure 1) for studying the effect of force on enzymatic proteolysis. We calibrated the tweezers for 1 μm and 3 μm beads using both the magnitude of the observed Brownian fluctuations and calculation of the roll-off frequency at varying magnet positions (Figure 2). In the force proteolysis experiments, the setup is similar except that the DNA is replaced with collagen (Figures 3, 4). The normalized number of beads remaining can be plotted as function of time to find the proteolysis rates (Figure 5), and this process can be repeated for varying enzyme concentrations and forces.
Figure 1. Schematic of the magnetic trap calibration process (not to scale). Two permanent rare earth magnets create a magnetic field that pulls on the superparamagnetic bead. Translating the magnet up and down adjusts the applied force. The beads are imaged using a conventional bright-field microscope with the light passing through a pinhole between the two magnets. Inset: Image taken with a 40x air objective. The sharp, round spots correspond to the beads attached to the coverslip surface. The out-of-focus objects are detached beads.
Figure 2. Plots of the calibrated force as a function of magnet distance from the sample surface for 1 μm (left) and 2.8 μm (right) beads. Data were fit to the empirical function , where x is the distance from the magnet. a = 31.8, b= 5.61, and c = 4.39 for the 1 μm beads and a = 140, b = 3.10 and c = 1.86 for the 3 μm beads. These values are specific to our specific instrument and experimental geometry, and each instrument should be calibrated individually. The error bars at each point represent a ~10% variability in applied force on a bead to bead basis, due to the variability in bead size. The force applied is proportional to the volume of the beads, and the bead volume varies ~9% over the mean (according to manufacturer specifications).
Figure 3. Force proteolysis assay setup (Not to scale). The collagen model trimer is attached to the surface of the coverslip via myc/anti-myc conjugation. The streptavidin-coated superparamagnetic beads are attached to the collagen trimers via a biotin-streptavidin linkage. Activated MMP-1 cuts the collagen over time, causing the beads to detach from the surface and move away from the focal plane.
Figure 4. Schematic cartoon of proteolysis as a function of time. The cartoon shows a sample field of view over time as proteolysis occurs. Over time, MMP-1 cuts the collagen and the beads detach and move away from the focal plane under the influence of the magnetic field.
Figure 5. Proteolysis rates depend on applied force16. Shown are data collected at 1.0 pN (3 μM MMP-1; black), 6.2 pN (3 μM MMP-1; red) and 13 pN (0.2 μM MMP-1; magenta). The rates of proteolysis (fit parameters) are: 0.22 ± 0.02 min-1 (1 pN), 0.46 ± 0.09 min-1 (6.2 pN), and 2.08 ± 0.18 min-1 (13 pN). The fraction of beads unproteolyzed at long time points (> 15 minutes) remains approximately constant at ~0.25 across different experiments. Error bars correspond to the Poisson statistics reflecting the number of observations at each time point. The error at each time point for n beads is n1/2. The error in fraction of beads attached was calculated by error propagation.1 μm beads were used for 1.0 pN and 6.2 pN experiments and 2.8 μm beads were used for 13 pN experiments.
This protocol describes a new use for a classical single molecule technique. Magnetic tweezers allow medium to high-throughput single molecule assays in a cost-efficient manner. However, like all experimental techniques there are challenges and potential pitfalls.
Limitations of magnetic tweezers
Compared to an optical trap the spatial and temporal resolution of a MT apparatus is low. Moreover, the forces generated by the simple MT described here are 30 pN or less, significantly less than forces routinely accessed in AFM experiments. This limitation can be a virtue: MT are well suited for applying sub-pN forces.
Unlike an optical trap or AFM, conventional MT, such as the apparatus here, do not allow the manipulation of individual particles. Optical traps are usually used to apply force parallel to the coverslip, while MT are best-suited for vertical pulling experiments. Electromagnetic MT address these limitations, although at the cost of increased complexity.
Both the Brownian fluctuation and power spectrum analysis for force calibration have their limitations. We found that the Brownian fluctuation method is particularly sensitive to camera blur. As the frame rate increased (exposure time decreased) the calculated force decreased. In practice, we increased frame rate until the forces calculated from Brownian fluctuations agreed with the forces calculated using the power spectrum to within 10%. Conversely, the power spectrum analysis is more robust against camera blur, but slightly more difficult to implement. We recommend performing both calculations to check for the robustness of the results.
We note that variations in bead size and magnetic particle content result in variations in applied force of ~10%. This effect was not important in our recent experiment because we average over hundreds of beads in each measurement. However, experiments that extract unique information from each tethered bead could potentially require a more accurate calibration method.
Controls to ensure single point attachments
Achieving specific, oriented, single-point attachments is often a practical challenge in single molecule studies. The following controls confirmed specific attachment of collagen to antibody, and beads to collagen:
In cases 1-5, where some component of the attachment series was deliberately omitted, anywhere from 0 to 4 beads were seen attached to the flow cell per field of view (80,000 μm2). Only in case 6, where all the attachment moieties are present, did we see ~30-60 2.8 μm beads and 80-200 1 μm beads attached to the surface. The proteolysis kinetics observed are adequately fit by a single exponential plus a constant term, which strongly suggests that there is only one rate limiting step, and hence only a single tether. The constant term likely reflects beads that are non-specifically attached to the coverslip.
We report the concentrations of antibody and protein that worked for our experiment. A wide range of concentrations for each component should be examined when developing a new assay. BSA worked well as a surface passivation agent in our experiment. However, this method may not work in all circumstances. Other protocols for surface passivation have also been used with success. Examples include polyethylene glycol functionalized glass slides17, supported lipid bilayers18, or casein as a blocking agent.
Notes on data analysis
In force proteolysis experiments, there is always some non-specific detachment (especially at higher forces19) due to dissociation of non-covalent (e.g. biotin - streptavidin) interactions. We account for this phenomenon by measuring proteolysis at various MMP-1 concentrations, including no MMP-1, as described in Adhikari et. al. Measuring proteolysis at various MMP-1 concentrations for all forces allows us to generate Michaelis-Menten curves, where are used to calculate the catalytic efficiency kcat/KM of the enzyme. Alternatively, if various concentrations of the enzyme are not used, we recommend that a control measurement be done with no enzyme at the given forces to subtract off the rate of non-specific detachment.
The magnetic tweezers cover a wide force range. The forces that can be exerted depend upon the size of the beads. 1 μm and 2.8 μm beads overlap in accessible forces, and in our experience, using either of the beads at the intermediate forces works well.
We anticipate that similar experimental approaches may be broadly applicable in studying force dependent proteolysis4,20, unfolding21 and binding interactions22. We and others23 have noted that the radius of diffraction rings for out of focus beads provides a sensitive means for tracking the position of the bead relative to the coverslip23. Although generally not used in this way, we believe that MT assays have the capacity to provide nanometer-precision measurements of protein structural dynamics. This additional information may be particularly valuable in the context of force-dependent proteolysis or binding measurements.
No conflicts of interest declared.
This work was supported by the Burroughs Wellcome Career Award at the Scientific Interface (A.R.D.), the National Institutes of Health through the NIH Director's New Innovator Award Program 1-DP2-OD007078 (A.R.D.), the William Bowes Jr. Stanford Graduate Fellowship (A.S.A.), and the Stanford Cardiovascular Institute Younger Predoctoral Fellowship (J.C.). The authors thank James Spudich for loaning microscopy equipment.
|Micro Cover Glass #1.5 (22x22)||VWR||48366-067|
|Micro Cover Glass #1.5 (22x40)||VWR||48393-048|
|T4 DNA Ligase||Invitrogen||15224-041|
|Microcon Ultracel YM-100||Millipore||42413|
|Bovine Serum Albumin||Sigma||B4287-5G|
|Dynabeads M-280 Streptavidin||Invitrogen||658.01D|
|Dynabeads MyOne T1 Streptavidin||Invitrogen||658.01D|
|Biotin labeled oligo||IDT DNA||Custom synthesis|
|Digoxigenin labeled oligo||IDT DNA||Custom synthesis|
|Collagen peptide gene||DNA 2.0||Custom synthesis|
|MMP-1 cDNA||Harvard Plasmid Database|
|Servo controller for translator||Thorlabs||TDC001|
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