Name: force spectroscopy of single protein molecules using an atomic force microscope
Uploaded: 2019-02-28T12:30:00
Description: Watch this Scientific Journal Video about Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope at JoVE.com

This work was supported by the National Science Foundation grants MCB-1244297 and MCB-1517245 to PEM.

1. Protein Preparation
<ol>
	<li>DNA cloning.
	<ol>
		<li>Synthesize a DNA sequence of interest, for example, the DNA sequence of NI10C10, or isolate via PCR from the host organism using standard molecular biology techniques11. Flank the gene of interest with restriction sites during synthesis or by placing sites in the 5&#8217;- end of the PCR primers to correspond to a module in the plasmid pEMI91 (Addgene #74888)12.</li>
		<l...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+single+molecule+force+spectroscopy+through+automated+real-time+data+collection+and+quantification+of+experimental+conditions.">Improving single molecule force spectroscopy through automated real-time data collection and quantification of experimental conditions.</a> Ultramicroscopy. 136, 7-14 (2014).</li><li>Bouchiat, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+persistence+length+of+a+worm-like+chain+molecule+from+force-extension+measurements.">Estimating the persistence length of a worm-like chain molecule from force-extension measurements.</a> Biophysical journal. 76 (1), 409-413 (1999).</li><li>Su, T., Purohit, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+forced+unfolding+of+proteins.">Mechanics of forced unfolding of proteins.</a> Acta. 5 (6), 1855-1863 (2009).</li><li>Steward, A., Toca-Herrera, J. L., Clarke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+cloning+system+for+construction+of+multimeric+proteins+for+use+in+atomic+force+microscopy.">Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy.</a> Protein science. 11 (9), 2179-2183 (2002).</li><li>Scholl, Z. N., Josephs, E. A., Marszalek, P. 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K., Hummer, G., Szabo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory,+analysis,+and+interpretation+of+single-molecule+force+spectroscopy+experiments.">Theory, analysis, and interpretation of single-molecule force spectroscopy experiments.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (41), 15755-15760 (2008).</li><li>Popa, I., Berkovich, R., Alegre-Cebollada, J., Rivas-Pardo, J. A., Fernandez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halotag+Tethers+to+Study+Titin+Folding+at+the+Single+Molecule+Level.">Halotag Tethers to Study Titin Folding at the Single Molecule Level.</a> Biophysical journal. 106 (2), 391a (2014).</li><li>Yu, H., Siewny, M. G., Edwards, D. T., Sanders, A. W., Perkins, T. 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A critical step in the protocol is the use of a polyprotein, described in step 1.1.2, which serves as a positive control to &#34;fingerprint&#34; single-molecule events. Generally, there must be unfolding events of the polyprotein proteins (for I91, this means an unfolding force of about 200 pN and contour length increment of about 28 nm) to unambiguously conclude that the protein of interest has been unfolded. For example, when the protein of interest is flanked by three I91 domains from either side, then there must be ...

Advances in single molecule force spectroscopy (SMFS) by AFM have enabled mechanical manipulation and precise characterization of single protein molecules. This characterization has produced novel insights about protein mechanics1,2, protein folding3, protein-ligand interactions4, protein-protein interactions5, and protein-based engineered materials6,7,8. SMFS is especially useful for studying protein unfolding, as stretching by AFM allows the chemical and physical bonds within the protein molecule to gradually extend according to their stiffness, which gives rise to a continually increasing contour length. This overstretching of a protein molecule can produce an abrupt transition in the force-extension curve resulting in a rupture event (or force peak). The force peak gives direct information on the unfolding force and structural change of the protein during the mechanical unfolding process. One of the first studies using AFM measured titin1 and found novel aspects of protein unfolding and refolding under physiological conditions without the use of unnatural denaturants like concentrated chemicals or extreme temperatures.
SMFS experiments are conducted on a variety of instruments, though here we consider only the AFM. The AFM is composed of four main elements: the probe, the detector, the sample holder, and the piezoelectric scanner. The probe is a sharp tip on the free-swinging end of a cantilever. After calibration, bending of the cantilever during stretching of an attached molecule is measured using a laser beam that is reflected off the back of the cantilever to precisely determine forces using Hooke&#39;s law. The reflected laser beam projects into a quadrant photodiode detector which produces a voltage in proportion to the displacement of the laser beam from the diode center. The substrate with the protein sample in fluid is mounted onto a 3D piezoelectric stage that can be controlled with sub-nanometer precision. A computer reads the voltage from the photodiode detectors and controls the 3D stage through a computer-controlled voltage supply. These piezo actuator stages are usually equipped with capacitive or strain-gauge position sensors to precisely measure piezo displacement and to correct hysteresis through feed-back control system. The sensor signal output from the piezo controller is converted into distance using the voltage constant of the piezo that is factory-calibrated. An example force-extension curve from a pulling experiment is shown in Figure 2.
There are two types of AFM-SMFS experiments: constant velocity and constant force pulling measurements. Constant force SMFS measurements are described in Oberhauser et al.9, while here we focus on constant velocity measurements. A typical AFM constant-velocity pulling experiment is done by providing voltage to a piezo to gently move a substrate relative to a cantilever tip. A typical experiment has the tip initially pressing against the surface. The pulling measurement is begun by moving the substrate away from the tip to bring out of contact. If a protein comes into contact with the tip initially, it will be pulled and the unfolding trace of force against displacement will be measured. The substrate is then brought back into contact with the tip and a relaxing trace is measured where protein folding can be determined from the force displacement.

<table>
	<tbody>
		<tr>
			<td>AFM Specimen Discs, 15mm diameter</td>
			<td>Ted Pella, Inc.</td>
			<td>16218</td>
			<td>Serve as base for glass substrate</td>
		</tr>
		<tr>
			<td>Round Glass Coverslips, 15mm diamiter No.1 Thick</td>
			<td>Ted Pella, Inc.</td>
			<td>26024</td>
			<td>serve as glass substrate and base for gold coating</td>
		</tr>
		<tr>
			<td>Adhesive Tabs</td>
			<td>Ted Pella, Inc.</td>
			<td>16079</td>
			<td>Paste on AFM Specimen Discs to provide a sticky face for attaching glass coverslips</td>
		</tr>
		<tr>
			<td>STD Multimode head assembly</td>
			<td>Bruker Nano Inc.</td>
			<td>1B75C</td>
			<td>AFM head</td>
		</tr>
		<tr>
			<td>Glass probe holder</td>
			<td>Bruker Nano Inc.</td>
			<td>MTFML-V2</td>
			<td>Glass probe holder for scanning in fluid with the MultiMode AFM.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Microlever AFM probes</td>
			<td>Bruker Nano Inc.</td>
			<td>MLCT</td>
			<td>Silicon Nitride cantilevers with Silicon Nitride tips, ideal for contact imaging modes</td>
		</tr>
		<tr>
			<td>AFM probes with Au coated tips</td>
			<td>Bruker Nano Inc.</td>
			<td>OBL-10</td>
			<td>Cantilevers for pulling on proteins with low unfolding force</td>
		</tr>
		<tr>
			<td>Multifunction Data Acquisition (DAQ) Card,16-Bit, 1 MS/s (Multichannel), 1.25 MS/s (1-Channel), 32 Analog Inputs</td>
			<td>National Instruments</td>
			<td>PCI-6259</td>
			<td>Data Acquisition for signals from AFM head and Piezo Actuators</td>
		</tr>
		<tr>
			<td>LISA Linear Piezo Stage Actuators</td>
			<td>Physik Instrumente LP</td>
			<td>P-753.11C</td>
			<td>Piezo Actuator to control the position of substrate and perform pulling measurements</td>
		</tr>
		<tr>
			<td>XY Piezo Stage</td>
			<td>Physik Instrumente LP</td>
			<td>P-541.2CD</td>
			<td>Piezo Actuator to control the position of substrate and scan on substrate surface</td>
		</tr>
	</tbody>
</table>

force spectroscopy of single protein molecules using an atomic force microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

Representative results from this protocol are shown in Figure 2. Both panels show representative force-extension curves from proteins. The top shows results from a I91 polyprotein, while the bottom shows the I91 protein flanking a protein-of-interest, the NI10C molecule. These recordings show the characteristic force of I91 (200 pN) and contour length increment (28 nm) which indicates that the alignment and calibration of the AFM was successful. Th...

Watch this Scientific Journal Video about Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope at JoVE.com

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

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.

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