Name: oaaep1-mediated enzymatic synthesis and immobilization of polymerized protein for single-molecule force spectroscopy
Uploaded: 2020-02-05T14:30:00.000+00:00
Duration: 8 min 34 s
Description: Watch this Scientific Journal Video about OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy at JoVE.com

This work was supported by the National Natural Science Foundation of China (Grant No. 21771103, 21977047), Natural Science Foundation of Jiangsu Province (Grant No. BK20160639) and Shuangchuang Program of Jiangsu Province.

1. Protein production
<ol>
	<li>Gene clone
	<ol>
		<li>Purchase genes coding for the protein of interest (POI): Ubiquitin, Rubredoxin (RD)51, the cellulose-binding module (CBM), dockerin-X domain (XDoc) and cohesion from Ruminococcus flavefacience, tobacco etch virus (TEV) protease, elastin-like polypeptides (ELPs).</li>
		<li>Perform polymerase chain reaction and use three-restriction digestion enzyme system BamHI-BglII-KpnI for recombining the gene from different protein fragments.</li>
		<li>Confirm all genes by direct DNA sequencing.</li>
	</ol>
	</li>
	<li>Proteins expression and production
	<ol>
		<li>Transform E. coli BL21(DE3) with the pQE80L-POI or pET28a-POI plasmid for expression.</li>
		<li>Pick one single colony into 15 mL of LB medium with respective antibiotics (e.g., 100 &#956;g/mL ampicillin sodium salt or 50 &#956;g/mL, kanamycin). Keep shaking the cultures at 200 rpm at 37 &#176;C for 16-20 h.</li>
		<li>Dilute the overnight cultures into 800 mL of LB medium (1:50 dilution). For rubredoxin, centrifuge the culture at 1,800 x g, then resuspend in 15 mL of M9 medium (supplemented with 0.4% glucose, 0.1 mM CaCl2, 2 mM MgSO4), and then dilute it into 800 mL of M9 medium.</li>
		<li>Incubate the culture at 37 &#176;C while shaking at 200 rpm, until the culture reaches an optical density at 600 nm (OD600) of 0.6. Save 100 &#956;L sample of the culture as the pre-induction control for testing protein expression.</li>
		<li>Induce protein expression by adding IPTG to a final concentration of 1 mM and shake the culture at 37 &#176;C for 4 h at 200 rpm. Reserve a 100 &#956;L sample of the culture as the post-induction control for testing protein expression.</li>
		<li>Centrifuge the culture at 13,000 x g for 25 min at 4 &#176;C and store at -80 &#176;C before purification. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Purification of protein of interest
	<ol>
		<li>Resuspend the cells in 25 mL of lysis buffer (50 mM Tris, 150 mM NaCl, pH 7.4 containing DNase, RNase, PMSF) and use a sonicator (15% amplitude) to lyse it for 30 min on ice.</li>
		<li>Clarify the cell lysate at 19,000 x g for 40 min at 4 &#176;C.</li>
		<li>Pack 1 mL (bed volume) of Co-NTA or Ni-NTA affinity column and wash the column with 10 column volumes (CV) of ultrapure water and then 10 CVs of wash buffer (50 mM Tris, 150 mM NaCl, 2 mM imidazole, pH 7.4) by gravity flow.</li>
		<li>Pass the protein supernatant through the column by gravity flow for three times.</li>
		<li>Pour wash buffer on the column with 50 CVs to move away contaminant proteins.</li>
		<li>Elute the bound protein with 3 CVs of ice-cold elution buffer (20 mM Tris, 400 mM NaCl, 250 mM imidazole, pH 7.4). When it comes to rubredoxin proteins, further anion exchange purification using anion exchange column at pH 8.5 at 4 &#176;C is necessary.</li>
		<li>Analyze the sample by SDS-PAGE.</li>
	</ol>
	</li>
</ol>
2. Functionalization of coverslip and cantilever surface
<ol>
	<li>Functionalized coverslip surface preparation
	<ol>
		<li>Dissolve 20 g of potassium chromate in 40 mL of ultrapure water. Slowly add 360 mL of concentrated sulfuric acid to the potassium chromate solution with glass rod stir gently and to prepare the chromic acid. 
		CAUTION: The chemical used here and the final chromic acid is strongly corrosive and acidic. Work with proper protective equipment. The solution releases heat when add concentrated sulfuric acid, which means slow adding and proper pause for cooling down.</li>
		<li>Clean and activate a glass coverslip at 80 &#176;C for 30 min by chromic acid treatment. Completely immerse the coverslips in 1% (v/v) APTES toluene solution for 1 h at room temperature while protecting them from light.</li>
		<li>Wash the coverslip with toluene and absolute ethyl alcohol and dry the coverslip with a stream of nitrogen.</li>
		<li>Incubate the coverslip at 80 &#176;C for 15 min and then cool down to room temperature.</li>
		<li>Add 200 &#956;L of sulfo-SMCC (1 mg/mL) in dimethyl sulfoxide (DMSO) solution between two immobilized coverslips and incubate for 1 h protected from light.</li>
		<li>Wash the coverslip with DMSO first and then with absolute ethyl alcohol to remove residual sulfo-SMCC.</li>
		<li>Dry the coverslip under a stream of nitrogen.</li>
		<li>Pipet 60 &#956;L of 200 &#956;M GL-ELP50nm-C protein solution onto a functionalized coverslip and incubate for about 3 h.</li>
		<li>Wash the coverslip with ultrapure water to remove the unreacted GL-ELP50nm-C. 
		NOTE: Functionalized coverslips are capable for about two weeks under storage at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Functionalized cantilever surface preparation
	<ol>
		<li>Clean the cantilevers at 80 &#176;C for 10 min by chromic acid treatment.</li>
		<li>Functionalize the cantilever by amino-silanization with 1% (v/v) APTES toluene solution and then bake the cantilever at 80 &#176;C for 15 min before conjugating to sulfo-SMCC.</li>
		<li>Link the C-ELP50nm-NGL to the surface with the maleimide group of sulfo-SMCC for 1.5 h.</li>
		<li>Wash away the unreacted C-ELP50nm-NGL on the coverslip by ultrapure water.</li>
		<li>Immerse a functionalized cantilever in 200 &#956;L of 50 &#956;M GL-CBM-XDoc protein solution containing 200 nM OaAEP1 at 25 &#176;C for 20-30 min. Then use AFM buffer (100 mM Tris, 100 mM NaCl, pH 7.4) to wash away unreacted protein. 
		NOTE: The surface chemistry of the cantilevers and the coverslip are similar.</li>
	</ol>
	</li>
</ol>
3. Stepwise polyprotein preparation with controlled sequences
<ol>
	<li>Link the ligation unit Coh-tev-L-POI-NGL to the GL-ELP50nm immobilized on the coverslip surface by OaAEP1 for 30 min.</li>
	<li>Use 15-20 mL of AFM buffer (100 mM Tris, 100 mM NaCl, pH 7.4) to wash away any unreacted proteins.</li>
	<li>Add 100 &#956;L of TEV protease (0.5 mM EDTA, 75 mM NaCl, 25 mM Tris-HCl 10% [v/v] glycerol, pH 8.0) to cleave the protein unit at the TEV recognize site for 1 h at 25 &#176;C.</li>
	<li>Use 15-20 mL of AFM buffer to wash away residual proteins.</li>
	<li>Link the ligation unit Coh-tev-L-POI-NGL to the GL-Ub-NGL-Glass by OaAEP1 for 30 min.</li>
	<li>Repeat steps 3.3 to 3.5 N-1 times to build protein construct GL-(Ub)n-NGL on the glass surface. Omit the last TEV cleavage reaction to reserve cohesin on the protein-polymer as Coh-tev-L-(Ub)n-NGL-Glass.</li>
</ol>
4. AFM Experiment measurement and data analysis
<ol>
	<li>AFM measurements
	<ol>
		<li>Add 1 mL of AFM buffer to the chamber with 10 mM CaCl2 and 5 mM Ascorbic Acid.</li>
		<li>Choose the D tip of the functionalized AFM probe for the experiment. Use the equipartition theorem to calibrate the cantilever in AFM buffer with an accurate spring constant (k) value before each experiment.</li>
		<li>Attach the cantilever tip to the sample surface to form the Cohesin/Dockerin pair.</li>
		<li>Retract the cantilever at a constant velocity of 400 nm&#183;s&#8722;1 from the surface. In the meantime, record the force-extension curve at a sample rate of 4000 Hz.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Use JPK data processing select force-extension traces.</li>
		<li>Use software to analyze the traces. Fit the curves with the worm-like-chain (WLC) model of polymer elasticity and obtain unfolding force and contour length increment for individual protein unfolding peak.</li>
		<li>Fit the histograms of unfolding forces with the Gaussian model to obtain the most probable values of unfolding force (&#60;Fu&#62;) and contour length increment (&#60;&#916;Lc&#62;).</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

We have described a protocol for enzymatic biosynthesis and immobilization of polyprotein and verified the polyprotein design by AFM-based SMFS. This methodology provides a novel approach to building the protein-polymer in a designed sequence, which complements previous methods for polyprotein engineering and immobilization4,6,52,53,54,<sup class="x...

Compared with synthetic polymers, natural multi-domain proteins have a uniform structure with a well-controlled number and type of subdomains1. This feature usually leads to improved biological function and stability2,3. Many approaches, such as cysteine-based disulfide bond coupling and recombinant DNA technology, have been developed for building such a polymerized protein with multiple domains4,5,6,7. However, the former method always results in a random and uncontrolled sequence, and the latter one leads to other problems, including the difficulty for the overexpression of toxic and large-size proteins and the purification of complex protein with cofactor and other delicate enzymes.
To meet this challenge, we develop an enzymatic method that conjugates protein monomer together for polymer/polyprotein in a stepwise fashion using a protein ligase OaAEP1 combined with a protease TEV8,9. OaAEP1 is a strict and efficient endopeptidase. Two proteins can be linked covalently as Asn-Gly-Leu sequence (NGL) through two termini by OaAEP1 in less than 30 min if the N-terminus is Gly-Leu residues(GL) and the other of which the C-terminus is NGL residues10. However, the use of OaAEP1 only to link protein monomer leads to a protein polymer with an uncontrolled sequence like the cysteine-based coupling method. Therefore, we design the N-terminus of the protein unit with a removable TEV protease site plus a leucine residue as ENLYFQ/G-L-POI. Before the TEV cleavage, the N-terminal would not participate in OaAEP1 ligation. And then the GL residues at N-terminus, which are compatible with further OaAEP1 ligation, is exposed after the TEV cleavage. Thus, we have achieved a sequential enzymatic biosynthesis method of polyprotein with a relatively well-controlled sequence.
Here, our stepwise enzymatic synthesis method can be used in polyprotein sample preparation, including sequence-controlled and uncontrolled, and protein immobilization for single-molecule studies as well, especially for the complex system such as metalloprotein.
Moreover, AFM-based SMFS experiments allow us to confirm the protein polymer construction and stability at the single-molecule level. Single-molecule force spectroscopy, including AFM, optical tweezer and magnetic tweezer, is a general tool in nanotechnology to manipulate biomolecule mechanically and measure their stability11,12,13,14,15,16,17,18,19,20. Single-molecule AFM has been widely used in the study of protein (un)folding21,22,23,24,25, the strength measurement of receptor-ligand interaction26,27,28,29,30,31,32,33,34,35, inorganic chemical bond20,36,37,38,39,40,41,42,43 and metal-ligand bond in metalloprotein44,45,46,47,48,49,50. Here, single-molecule AFM is used to verify the synthesized polyprotein sequence based on the corresponding protein unfolding signal.

<table><tbody><tr><td>iron (III) chloride hexahydrate</td><td>Energy chemical</td><td></td><td>99%</td></tr><tr><td>Zinc chloride</td><td>Alfa Aesar</td><td></td><td>100.00%</td></tr><tr><td>calcium chloride hydrate</td><td>Alfa Aesar</td><td></td><td>99.9965% crystalline aggregate</td></tr><tr><td>L-Ascorbic Acid</td><td>Sigma Life Science</td><td></td><td>Bio Xtra, &amp;ge;99.0%, crystalline</td></tr><tr><td>(3-Aminopropyl) triethoxysilane</td><td>Sigma-Aldrich</td><td></td><td>&amp;ge;99%</td></tr><tr><td>sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate</td><td>Thermo Scientific</td><td></td><td>90%</td></tr><tr><td>Glycerol</td><td>Macklin</td><td></td><td>99%</td></tr><tr><td>5,5&#039;-dithiobis(2-nitrobenzoic acid)</td><td>Alfa Aesar</td><td></td><td></td></tr><tr><td>Genes</td><td>Genscript</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nanowizard 4 AFM</td><td>JPK Germany</td><td></td><td></td></tr><tr><td>MLCT cantilever</td><td>Bruker Corp</td><td></td><td></td></tr><tr><td>Mono Q 5/50 GL</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>AKTA FPLC system</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Glass coverslip</td><td>Sail Brand</td><td></td><td></td></tr><tr><td>Nanodrop 2000</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Avanti JXN-30 Centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Gel Image System</td><td>Tanon</td><td></td><td></td></tr></tbody></table>

oaaep1-mediated enzymatic synthesis and immobilization of polymerized protein for single-molecule force spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

The NGL residues introduced between adjacent proteins by OaAEP1 ligation will not affect protein monomer stability in the polymer as the unfolding force (&#60;Fu&#62;), and contour length increment (&#60;&#916;Lc&#62;) is comparable with the previous study (Figure 1). The purification result of the rubredoxin protein is shown in Figure 2. To prove the protein after TEV cleavage is compatible with the following OaAEP1 ligation to construct p...

Watch this Scientific Journal Video about OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy at JoVE.com

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

oaaep1-mediated-enzymatic-synthesis-immobilization-polymerized

Research

JoVE Journal

Biochemistry

6.7K Views.  Nanjing University. Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Video: OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

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 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.

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.

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

Bioengineering

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.

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. ...

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

Engineering

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Chemistry

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.

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 ...

Force-Clamp Rheometry for Characterizing Protein-based Hydrogels

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog proportional-integral-derivative (PID) system to apply controlled-force protocols on cylindrical protein-based hydrogel samples, which are tethered between a linear voice-coil motor and a force transducer. During operation, the PID system adjusts the extension of the hydrogel sample to follow a predefined force protocol by minimizing the difference between the measured and set-point forces. This unique approach to protein-based hydrogels&#160;enables the tethering of extremely low-volume hydrogel samples (&#60; 5 &#181;L) with different protein concentrations. Under force-ramp protocols, where the applied stress increases and decreases linearly with time, the system enables the study of the elasticity and hysteresis behaviors associated with the (un)folding of proteins and the measurement of standard elastic and viscoelastic parameters. Under constant-force, where the force pulse has a step-like shape, the elastic response, due to the change in force, is decoupled from the viscoelastic response, which comes from protein domain unfolding and refolding. Due to its low-volume sample and versatility in applying various mechanical perturbations, force-clamp rheometry is optimized to investigate the mechanical response of proteins under force using a bulk approach.

A new force-clamp rheometry technique is used to investigate the mechanical properties of low-volume protein-based hydrogel samples tethered between a voice-coil motor and a force sensor. An analog proportional-integral-derivative (PID) system allows for the &#39;clamping&#39; of the force experienced to the desired protocol.

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog ...

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping.
These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments.
These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...