Name: investigating receptor-ligand systems of the cellulosome with afm-based single-molecule force spectroscopy
Uploaded: 2013-12-20T13:45:00
Description: Watch this Scientific Journal Video about Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy at JoVE.com

The authors acknowledge funding from a European Research Council advanced grant to Hermann Gaub. Michael A. Nash gratefully acknowledges funding from Society in Science - The Branco Weiss Fellowship program. The authors thank Edward A. Bayer, Yoav Barak, and Daniel B. Fried at the Weizmann Institute of Science for generously providing the proteins used in this study. The authors thank Hermann E. Gaub, Elias M. Puchner, and Stefan W. Stahl for helpful discussions.

A schematic of the pulling geometry used in this work to probe the cohesin-dockerin interaction is shown in Figure 1A. The protein immobilization protocol reported here for cantilever and cover glass functionalization is a modified version of the procedure published previously27. The proteins were expressed from plasmid vectors in E. coli using conventional methods. The proteins were designed with a solvent-accessible thiol group, which was used in combination with maleimide chemistry...

<ol>
	<li>Bayer, E. A., Belaich, J. P., Shoham, Y., Lamed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellulosomes:+Multienzyme+machines+for+degradation+of+plant+cell+wall+polysaccharides.">The cellulosomes: Multienzyme machines for degradation of plant cell wall polysaccharides.</a> Annu. Rev. Microbiol. 58, 521-554 (2004).</li><li>Bayer, E. A., Lamed, R., White, B. A., Flint, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Cellulosomes+to+Cellulosomics.">From Cellulosomes to Cellulosomics.</a> Chem. Rec. 8 (6), 364-377 (2008).</li><li>Binnig, G., Quate, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscope.">Atomic Force Microscope.</a> Phys. Rev. Lett. 56 (9), 930-933 (1986).</li><li>Garc&#305;&#769;a, R., Perez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+atomic+force+microscopy+methods.">Dynamic atomic force microscopy methods.</a> Surf. Sci. Rep. 47 (6), 197-301 (2002).</li><li>Engel, A., M&#252;ller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observing+single+biomolecules+at+work+with+the+atomic+force+microscope.">Observing single biomolecules at work with the atomic force microscope.</a> Nat. Struct. Biol. 7 (9), 715-718 (2000).</li><li>Li, H., Cao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Mechanics:+From+Single+Molecules+to+Functional+Biomaterials.">Protein Mechanics: From Single Molecules to Functional Biomaterials.</a> Acc. Chem. Res. 43 (10), 1331-1341 (2010).</li><li>Florin, E. -. L., Moy, V. T., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+forces+between+individual+ligand-receptor+pairs.">Adhesion forces between individual ligand-receptor pairs.</a> Science. 264 (5157), 415-417 (1994).</li><li>Oberhauser, A., Hansma, P., Carrion-Vazquez, M., Fernandez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+unfolding+of+titin+under+force-clamp+atomic+force+microscopy.">Stepwise unfolding of titin under force-clamp atomic force microscopy.</a> Proc. Natl. Acad. Sci. U.S.A. 98 (2), 468-472 (2001).</li><li>Puchner, E. M., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+function:+probing+proteins+with+AFM-based+force+spectroscopy.">Force and function: probing proteins with AFM-based force spectroscopy.</a> Curr. Opin. Struct. Biol. 19 (5), 605-614 (2009).</li><li>Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J., Gaub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+unfolding+of+individual+titin+immunoglobulin+domains+by+AFM.">Reversible unfolding of individual titin immunoglobulin domains by AFM.</a> Sci. 276 (5315), 1109-1112 (1997).</li><li>Stahl, S. W., Nash, M. A., 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=Single-molecule+dissection+of+the+high-affinity+cohesin&#8211;dockerin+complex.">Single-molecule dissection of the high-affinity cohesin&#8211;dockerin complex.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (50), 20431-20436 (2012).</li><li>Boland, T., Ratner, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+hydrogen+bonding+in+DNA+nucleotide+bases+by+atomic+force+microscopy.">Direct measurement of hydrogen bonding in DNA nucleotide bases by atomic force microscopy.</a> Proc. Natl. Acad. Sci. U.S.A. 92 (12), 5297-5301 (1995).</li><li>Morfill, J., K&#252;hner, F., Blank, K., Lugmaier, R. A., Sedlmair, J., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-S+Transition+in+Short+Oligonucleotides.">B-S Transition in Short Oligonucleotides.</a> Biophys. J. 93 (7), 2400-2409 (2007).</li><li>Rief, M., Clausen-Schaumann, H., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-dependent+mechanics+of+single+DNA+molecules.">Sequence-dependent mechanics of single DNA molecules.</a> Nat. Struct. Biol. 6 (4), 346-350 (1999).</li><li>Severin, P. M. D., Zou, X., Gaub, H. E., Schulten, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytosine+methylation+alters+DNA+mechanical+properties.">Cytosine methylation alters DNA mechanical properties.</a> Nucleic Acids Res. 39 (20), 8740-8751 (2011).</li><li>Geisler, M., Balzer, B. N., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+Adhesion+at+the+Solid-Liquid+Interface+Probed+by+a+Single-Molecule+Force+Sensor.">Polymer Adhesion at the Solid-Liquid Interface Probed by a Single-Molecule Force Sensor.</a> Small. 5 (24), 2864-2869 (2009).</li><li>Giannotti, M. I., Vancso, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogation+of+Single+Synthetic+Polymer+Chains+and+Polysaccharides+by+AFM-Based+Force+Spectroscopy.">Interrogation of Single Synthetic Polymer Chains and Polysaccharides by AFM-Based Force Spectroscopy.</a> Chem. Phys. Chem. 8 (16), 2290-2307 (2007).</li><li>Hugel, T., Rief, M., Seitz, M., Gaub, H., Netz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Stretched+Single+Polymers:+Atomic-Force-Microscope+Experiments+Versus+Ab-Initio+Theory.">Highly Stretched Single Polymers: Atomic-Force-Microscope Experiments Versus Ab-Initio Theory.</a> Phys. Rev. Lett. 94 (4), 10-1103 (2005).</li><li>Nash, M. A., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Molecule+Adhesion+of+a+Stimuli-Responsive+Oligo(ethylene+glycol)+Copolymer+to+Gold.">Single-Molecule Adhesion of a Stimuli-Responsive Oligo(ethylene glycol) Copolymer to Gold.</a> ACS Nano. 6 (12), 10735-10742 (2012).</li><li>Merkel, R., Nassoy, P., Leung, A., Ritchie, K., Evans, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+landscapes+of+receptor-ligand+bonds+explored+with+dynamic+force+spectroscopy.">Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy.</a> Nature. 397 (6714), 50-53 (1999).</li><li>Morfill, J., Neumann, J., 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=Force-based+Analysis+of+Multidimensional+Energy+Landscapes:+Application+of+Dynamic+Force+Spectroscopy+and+Steered+Molecular+Dynamics+Simulations+to+an+Antibody+Fragment&#8211;Peptide.">Force-based Analysis of Multidimensional Energy Landscapes: Application of Dynamic Force Spectroscopy and Steered Molecular Dynamics Simulations to an Antibody Fragment&#8211;Peptide.</a> Complex. J. Mol. Biol. 381 (5), 1253-1266 (2008).</li><li>Ortiz, C., Hadziioannou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entropic+Elasticity+of+Single+Polymer+Chains+of+Poly(methacrylic+acid)+Measured+by+Atomic+Force+Microscopy.">Entropic Elasticity of Single Polymer Chains of Poly(methacrylic acid) Measured by Atomic Force Microscopy.</a> Macromolecules. 32 (3), 780-787 (1999).</li><li>Bustamante, C., Marko, J. F., Siggia, E. D., Smith, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entropic+Elasticity+of+l-phage+DNA.">Entropic Elasticity of l-phage DNA.</a> Science. 265 (5178), 1599-1600 (1994).</li><li>Marko, J. F., Siggia, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+DNA.">Stretching DNA.</a> Macromolecules. 28 (26), 8759-8770 (1995).</li><li>Puchner, E. M., Franzen, G., Gautel, M., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+Proteins+by+Their+Unfolding+Pattern.">Comparing Proteins by Their Unfolding Pattern.</a> Biophys. J. 95 (1), 426-434 (2008).</li><li>Livadaru, L., Netz, R. R., Kreuzer, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+Response+of+Discrete+Semiflexible+Polymers.">Stretching Response of Discrete Semiflexible Polymers.</a> Macromolecules. 36 (10), 3732-3744 (2003).</li><li>Zimmermann, J. L., Nicolaus, T., Neuert, G., Blank, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol-based,+site-specific+and+covalent+immobilization+of+biomolecules+for+single-molecule+experiments.">Thiol-based, site-specific and covalent immobilization of biomolecules for single-molecule experiments.</a> Nat. Protoc. 5 (6), 975-985 (2010).</li><li>Gumpp, H., Stahl, S. W., Strackharn, M., Puchner, E. M., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastable+combined+atomic+force+and+total+internal+fluorescence+microscope.">Ultrastable combined atomic force and total internal fluorescence microscope.</a> Rev. Sci. Instrum. 80 (6), 063704 (2009).</li><li>Gustafsson, M. G. 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=Scanning+force+microscope+springs+optimized+for+optical-beam+deflection+and+with+tips+made+by+controlled+fracture.">Scanning force microscope springs optimized for optical-beam deflection and with tips made by controlled fracture.</a> J. Appl. Phys. 76 (1), 172 (1994).</li><li>Cook, S. M., Lang, K. M., Chynoweth, K. M., Wigton, M., Simmonds, R. W., Sch&#228;ffer, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+implementation+of+dynamic+methods+for+measuring+atomic+force+microscope+cantilever+spring+constants.">Practical implementation of dynamic methods for measuring atomic force microscope cantilever spring constants.</a> Nanotechnology. 17 (9), 2135-2145 (2006).</li><li>Proksch, R., Sch&#228;ffer, T. E., Cleveland, J. P., Callahan, R. C., Viani, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finite+optical+spot+size+and+position+corrections+in+thermal+spring+constant+calibration.">Finite optical spot size and position corrections in thermal spring constant calibration.</a> Nanotechnology. 15 (9), 1344-1350 (2004).</li><li>Ainavarapu, S. R. K., Brujic, J., 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=Contour+Length+and+Refolding+Rate+of+a+Small+Protein+Controlled+by+Engineered+Disulfide+Bonds.">Contour Length and Refolding Rate of a Small Protein Controlled by Engineered Disulfide Bonds.</a> Biophys. J. 92 (1), 225-233 (2007).</li><li>Dietz, H., Rief, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+Molecular+Fingerprints+in+Single+Molecule+Force+Spectroscopy+Using+Pattern+Recognition.">Detecting Molecular Fingerprints in Single Molecule Force Spectroscopy Using Pattern Recognition.</a> Jap. J. Appl. Phys. 46, 5540-5542 (2007).</li><li>Evans, E., Ritchie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+strength+of+molecular+adhesion+bonds.">Dynamic strength of molecular adhesion bonds.</a> Biophys. J. 72 (4), 1541-1555 (1997).</li><li>Dietz, H., Rief, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+structure+by+mechanical+triangulation.">Protein structure by mechanical triangulation.</a> Proc. Natl. Acad. Sci. U.S.A. 103 (5), 1244-1247 (2006).</li><li>Dudko, O. 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=Intrinsic+Rates+and+Activation+Free+Energies+from+Single-Molecule+Pulling+Experiments.">Intrinsic Rates and Activation Free Energies from Single-Molecule Pulling Experiments.</a> Phys. Rev. Lett. 96 (10), 108101 (2006).</li><li>Friddle, R. W., Noy, A., De Yoreo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+the+widespread+nonlinear+force+spectra+of+intermolecular+bonds.">Interpreting the widespread nonlinear force spectra of intermolecular bonds.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (34), 13573-13578 (2012).</li></ol>

To obtain meaningful data from single molecule force spectroscopy experiments, it is crucial to achieve well-defined and reproducible pulling geometries. The protocol used here results in site-specific immobilization of protein complexes in a defined pulling geometry.
The cantilevers used in this study were chosen due to their force sensitivity and high resonance frequency in water. Moreover, the small tip curvature of approximately 10 nm is advantageous for single molecule experiments due to ...

Cellulosomes are large multienzyme complexes displayed on the surface of anaerobic cellulolytic bacteria (e.g.&#160;C. thermocellum) that have evolved to efficiently depolymerize plant cell wall lignocellulose into soluble oligosaccharides1. A central attribute of cellulosomes is the high-affinity cohesin-dockerin interaction. In the most prominent paradigm, a highly conserved 60-75 amino acid type I dockerin module is displayed at the C-terminal end of the various bacterial enzymes. The dockerin module directs assembly of synergistic combinations of enzymes onto the noncatalytic scaffold protein (&#39;scaffoldin&#39;), which comprises a polyprotein of cohesin domains that are specific for the type I dockerin module. At higher levels, cellulosome architecture can become very complex, incorporating alternative cohesin and dockerin pairs (e.g.&#160;type II, type III) that anchor the structures to the cell surface and allow for the assembly of branched structures containing multiple scaffoldins2. The various cohesin-dockerin types, despite having related structures, exhibit differential binding specificities suppressing cross reactivity with unintended scaffoldins or components from other cellulosome-producing bacterial species. While bioinformatic approaches have successfully identified thousands of unique cellulosomal components at the genetic level, comparatively few protein structures are known, and the mechanisms at work in cohesin-dockerin specificity determination remains an active area of structural biology research.
Since the invention of the atomic force microscope (AFM) by Binnig et al.3, various AFM operational modes have been developed and continuously improved, including noncontact imaging, oscillation mode imaging4, and single molecule force spectroscopy (SMFS)5,6. SMFS has evolved into a widely used technique to directly probe individual proteins7-11, nucleic acids12-15, and synthetic polymers16-19. In a typical SMFS experiment to investigate receptor-ligand binding20,21, an AFM cantilever tip is modified with one of the binding partners, while a flat glass surface is modified with the complementary binding partner. The modified cantilever is brought into contact with the surface allowing the partners to bind. The base of the cantilever is then withdrawn at constant speed and the force is measured using the optical lever deflection method. The resultant force-distance data traces exhibit sawtooth-like peaks if binding was established. In cases where the binding partners are fused to multiple protein domains, each peak in the force-distance trace can be correlated to the unfolding of a single protein domain or folded subdomain, while the last peak corresponds to rupture of the protein binding interface. The specific positions of the force-resistant elements can be used as a fingerprint to identify the various protein domains of interest. This method can be used to interrogate important amino acids involved in protein folding and stabilization. Many models have been reported in the literature to treat the characteristic force extension behavior observed in SMFS experiments. The most commonly used models include the freely jointed chain (FJC) model22, the worm-like chain (WLC) model18,23-25, and the freely rotating chain (FRC) model25,26.
In our prior work11, we used single-molecule force spectroscopy to investigate the interaction of cohesin and dockerin modules. Here, we present an experimental protocol for glass surface and cantilever functionalization with enzyme-dockerin and CBM-cohesin protein constructs. We also present an AFM-based SMFS protocol including data acquisition and analysis procedures. The described protocol can easily be generalized to other molecular systems, and should prove particularly useful to researchers interested in high-affinity receptor ligand pairs.

<table border="0" cellpadding="0" cellspacing="0" width="641">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3-Aminopropyl dimethyl ethoxysilane</td>
			<td style="width:139px;">ABCR GmbH</td>
			<td style="width:119px;">AB110423</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5 kDa NHS-PEG-maleimide</td>
			<td style="width:139px;">Rapp Polymer</td>
			<td style="width:119px;">13 5000-65-35</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TCEP Disulfide reducing gel</td>
			<td style="width:139px;">Thermo Scientific, Pierce</td>
			<td align="right" style="width:119px;">77712</td>
			<td><a href="http://www.thermoscientific.com/pierce">www.thermoscientific.com/pierce</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris(hydroxymethyl)aminomethane</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BioLever mini silicon nitride cantilevers</td>
			<td style="width:139px;">Olympus</td>
			<td style="width:119px;">BL-AC40TS-C2</td>
			<td>Soft batches</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">XYZ Piezoelectric actuators</td>
			<td style="width:139px;">Physik Instrumente GmbH</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Infrared &#8220;broad spectrum&#8221; IR laser</td>
			<td style="width:139px;">Superlum</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MFP-3D AFM Controller</td>
			<td style="width:139px;">Asylum Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Igor Pro 6.31</td>
			<td style="width:139px;">Wavemetrics</td>
			<td style="width:119px;"></td>
			<td>Data acquisition and analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pH Meter</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium borate</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tweezers</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cover glasses</td>
			<td style="width:139px;">Thermo Scientific, Menzel-Gl&#228;ser</td>
			<td style="width:119px;"></td>
			<td>24 mm diameter, 0.5 mm thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PTFE sample holder</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>custom made</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sonicator bath</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>analytical purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sulfuric acid (concentrated)</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>analytical purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide (30%)</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>analytical purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Orbital shaker</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Toluene</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>analytical purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Filter paper</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass slides</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microtubes</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micropipettes</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>suitable for microtubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rotator</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dishes</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beakers</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Optical microscope</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating receptor-ligand systems of the cellulosome with afm-based single-molecule force spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

We used the described procedure to investigate a type I cohesin-dockerin pair from C. thermocellum. Upon successful binding of the cohesin-dockerin pair, the recorded force distance traces showed characteristic peak patterns. A typical trace is shown in Figure 4a. Every peak in the trace represents the unfolding of one protein subdomain with the last peak corresponding to the dissociation of the receptor-ligand complex.
For the CBM-cohesin-dockerin-xylanase complex in...

Watch this Scientific Journal Video about Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy at JoVE.com

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Research

JoVE Journal

Bioengineering

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Single-molecule imaging has greatly advanced our understanding of molecular mechanisms in biological studies. However, it has been challenging to obtain ...

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...