This work was supported by the Marie Curie International Reintegration Grant (EP7). P. D. acknowledges the support of the Israel Council for Higher Education.

1. Tip Modification
<ol>
	<li>Purchase silicon nitride (Si3N4) AFM cantilevers with silicon tips (nominal cantilever radius of ~2 nm).</li>
	<li>Clean each AFM cantilever by dipping in anhydrous ethanol for 20 min. Dry at room temperature. Then treat the cantilevers by exposing them to O2 plasma for 5 min.</li>
	<li>Suspend the clean tips above (3 cm) a solution containing methyltriethoxysilane and 3-(aminopropyl) triethoxysilane in a ratio of 15:1 (v/v) in a desiccator under an inert atmosphere (either nitrogen or argon) and connect the desiccator to a vacuum pump. Vacuum for 2 h to form a monolayer of these two types of mixed silane compounds.</li>
	<li>Use a metallic tip holder (fabricated for this process) to place the tips on a hot plate. Then dry the tips for 10 minutes at 70 &#176;C under atmospheric conditions. Before use, clean the hot plate, metallic holder and tweezers using ethanol.</li>
	<li>Cool the tips at room temperature, and then immerse the tips into a solution of Fluorenylmethyloxycarbonyl-PEG-N-hydroxysuccinimide (Fmoc-PEG-NHS, MW 5,000 Da) at a concentration of 5 mM in chloroform containing 0.5% (v/v) triethylamine for 1 h at room temperature.</li>
	<li>Dip the tips in chloroform for 5 min and then dip it in dimethylformamide (DMF) for additional 5 min. In order to deprotect the Fmoc group of the attached PEG molecules, dip the tips in 20% piperidine (v/v) in DMF for 30 min. Dip the tips in DMF for 4 min and then in N-methyl-2-pyrrolidone (NMP) for additional 4 min. Repeat the sequential dipping three times.</li>
	<li>For coupling of amino acids, immerse the tips into a solution containing N-terminal protected amino acid (AA) / diisopropylethylamine (DIPEA) / 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluophosphate (HBTU), in a molar ratio of 1:1:1 at a total concentration of 30 mM in 5 mL NMP for 1.5 h.</li>
	<li>For peptide coupling, dip the tips into a 5 mL solution containing 40 mg of the protected peptide (N terminal and side chains, for example Fmoc-Gln(Trt)-Pro-Ala-Ser(tBu)-Ser(tBu)-Arg(pbf)-Tyr(tBu)-COOH.), 15 mg 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and 5 mL of DIPEA in NMP for 2 h.</li>
	<li>Dip the tips in NMP for 4 min. Then, to protect the reaming free/unreacted NH2 by the acetyl group, dip the tips for 30 min in a mixture of acetic anhydride/DIPEA at a molar ratio 4:1 and a total concentration of 0.65 M in NMP.</li>
	<li>For peptide coupling, perform two additional steps.
	<ol>
		<li>In order to deprotect the side chains of the peptide, immerse the tips into a solution containing 95% TFA, 2.5% triisopropylsilane, and 2.5% water for 1 h, and then wash with chloroform and DMF.</li>
		<li>In order to remove the Fmoc group of the peptide, immerse the tips into 20% piperidine (v/v) in DMF for 30 min.</li>
	</ol>
	</li>
	<li>Sequentially dip the peptide/amino acid-functionalized tips for four min each in DMF (for peptides) or NMP (for amino acids), chloroform, 50% ethanol and water. Finally dry the tip in air.</li>
</ol>
2. Surface Preparation
<ol>
	<li>Prepare mica. Cleave the substrates (9.9 mm diameter) before each use by using scotch tape. Then, wash the surfaces with triple distilled water (TDW).</li>
	<li>Prepare TiO2-coated silicon.
	<ol>
		<li>Cut the silicon wafer (100) into 2 cm squares using a diamond pen.</li>
		<li>Place the substrate in a 15 mL test tube filled with acetone and sonicate it for five minutes in an ultrasonic bath. Then, place this surface in a 15 mL test tube filled with isopropanol and sonicate it for 5 min. Dry the substrate using nitrogen.</li>
		<li>Dissolve the surfactant (e.g., Byk-348) in ethanol to prepare a 5% (weight/volume) solution. Then, add 0.02 mL of the surfactant solution to a 2 mL of 30% TiO2 dispersion.</li>
		<li>From the resulting solution, drop-cast 0.2 mL on a clean Si substrate.</li>
		<li>Anneal these drop-casted surfaces at 250 &#176;C for 2 h in air.41</li>
	</ol>
	</li>
</ol>
3. Single-molecule Force Spectroscopy Measurements
<ol>
	<li>Attach the desired surface to a metallic holder of the AFM with commercially available two-component glue. Place the metallic holder in the glass holder of the AFM, which is shaped as a small Petri&#160;dish. Fill this holder with Tris buffer (50 mM pH = 7.4) or any desired medium. Then, place the holder on the AFM stage below the tip holder.</li>
	<li>Calibrate the AFM cantilevers with spring constants ranging from 10 to 30 pN/nm using the thermal fluctuation method26 (included in the AFM software) with an absolute uncertainty of about 10%.</li>
	<li>Measure the force of the interaction by approaching the amino acid or peptide-functionalized tip to the substrate until it is in contact with the substrate with a compression force of ~200 pN and then immediately retract the tip at various speeds, from 0.1 to 0.8 &#181;m/s, for a distance of ~200 nm.</li>
</ol>
4. Data Analysis
<ol>
	<li>Convert the deflection values (V) to force by multiplying the photodiode sensitivity (V/m) and using the experimentally determined spring constant.42 This is done automatically by the AFM software.
	<ol>
		<li>To obtain F-D curves with one single molecule events, record several hundreds of curves (800-1,500). Obtain two peaks in a single-molecule curve: the first peak indicates nonspecific interactions between the tip and the surface and the second peak corresponds to the specific interaction of the molecule with the surface. When, the F-D curve contain more than these two peaks, discard them from the calculation of the most probable force. 
		NOTE: Only single adhesion events are taken into an account (from 10 to 30% of the curves).43</li>
	</ol>
	</li>
	<li>To calculate the apparent loading rate, fit at least 50 force vs. distance curves with the worm-like chain (WLC) model44 just prior to the rupture to obtain a set of loading rates, which are then used for preparing histograms of the apparent loading rates. Derive the unbinding forces between the peptides/amino acids and surface from the jump in force after separating the cantilever from the substrate.</li>
</ol>

The authors declare that they have no competing financial interests.

Steps 1.3, 1.4 and 1.7 in the protocol should be carried out with extensive care and in a very gentle manner. In step 1.3, the tip should not be in contact with the silane mixture and the silanization process should be carried out in an inert atmosphere (moisture free).45 This is done in order to prevent multilayer formation and because silane molecules readily undergo hydrolysis in the presence of moisture.45
In step 1.4, the temperature and time should be kept properly. Before starting step 1.5, the tip should be cooled down to room temperature; otherwise it will be damaged. In the coupling step (1.7), the HBTU and the examined amino acid or peptide should be completely dissolved in the mixture. After coupling, washing the tip with the different solvents should be done in a very gentle manner to avoid any damage to the tip.
The reported technique can be applied to any peptide or amino acid. To modify the silicon tip, we use silanes. This is general chemistry which can be altered. For example, one can use either two or one type of PEGylated silane to modify the tip.23,25,26 If the tip is made of gold, then thiol groups can be used for the modification of the tip. Alternative protocols exploit nitrilotriacetate (NTA)-terminated linkers, able to target histidine, together with recombinant histidine-tagged proteins. Recently, Lyubchenko et al. described the synthesis and examining of a novel polymer linker and showed its application in several SMFS experiments. The synthesis of the linker is based on the well-developed phosphoramidate (PA) chemistry. This chemistry allows a routine synthesis of linkers with predetermined lengths and PA composition. These linkers are homogeneous in length and can be terminated with various functional groups. Furthermore, biomolecules can be anchored on gold substrates or tips through native or engineered thiol groups as gold form covalent bonds with the sulfur atoms.
This method allows the quantitative and detailed measurement of the force needed to unbind a molecule from a surface at the single-molecule level and not in bulk. Future applications of the technique include the attachment of proteins to the tip and the design of new hybrid materials. The design and development of new composite materials and functional surfaces will benefit from obtaining a fundamental understanding of the interactions between proteins or peptides with inorganic materials. The protocol provided here for SMFS with AFM can serve as a powerful tool for studying the interactions between proteins, peptides and amino acids with different surfaces.

The interaction between proteins and inorganic minerals leads to the construction of composite materials with distinctive properties. This includes materials with high mechanical strength or unique optical properties.1,2 For example, the combination of the protein collagen with the mineral hydroxyapatite generates either soft or hard bones for different functionalities. 3 Short peptides can also bind inorganic materials with high specificity. 4,5,6 The specificity of these peptides has been used for designing new magnetic and electronic materials,7,8,9 fabricating nanostructured materials, growing crystals, 10 and synthesizing nanoparticles.11Understanding the mechanism underlying interactions between peptides or proteins and inorganic materials will therefore allow us to design new composite materials with improved adsorptive properties. In addition, since the interphase of implants with an immune response is mediated by proteins, better understanding the interactions of proteins with inorganic materials will improve our ability to design implants. Another important area that involves proteins interacting with inorganic surfaces is the fabrication of antifouling materials.12,13,14,15 Biofouling is an undesirable process in which organisms attach to a surface. It has many detrimental implications on our lives. For example, biofouling of bacteria on medical devices leads to hospital-acquired infections. Biofouling of marine organisms on boats and large ships increases the consumption of fuel.12,16,17,18
Single-molecule force spectroscopy (SMFS), using an AFM, can directly measure the interactions between an amino acid or a peptide with a substrate.19,20,21,22,23,24,25,26 Other methods such as phage display,27,28 quartz crystal microbalance (QCM)29or surface plasmon resonance (SPR)29,30,31,32,33 measure the interactions of peptides and proteins to inorganic surfaces in bulk.34,35,36 This means that the results obtained by these methods relate to ensembles of molecules or aggregates. In SMFS, one or very few molecules are fixed to the AFM tip and their interactions with the desired substrate is measured. This approach can be expanded to study protein folding by pulling the protein from the surface. In addition, it can be used to measure interactions between cells and proteins and the binding of antibodies to their ligands.37,38,39,40 This paper describes in detail how to attach either peptides or amino acids to the AFM tip using silanol chemistry. In addition, the paper explains how to perform force measurements and how to analyze the results.

<table><tbody><tr><td>Silicon nitride (Si&lt;sub&gt;3&lt;/sub&gt;N&lt;sub&gt;4&lt;/sub&gt;) AFM cantilevers with silicon tips</td><td>Bruker (Camarilo, CA, USA)</td><td></td><td>MSNL10, nominal cantilevers radius ~2 nm&amp;nbsp;</td></tr><tr><td>Methyltriethoxysilane&amp;nbsp;</td><td>Acros Organics (New Jersey, USA)</td><td></td><td>For Silaylation of the AFM tip&amp;nbsp;</td></tr><tr><td>3-(Aminopropyl) triethoxysilane</td><td>Sigma-Aldrich (Jerusalem, Israel)</td><td></td><td>For Silaylation of the AFM tip</td></tr><tr><td>Triisopropylsilane</td><td>Sigma-Aldrich (Jerusalem, Israel)</td><td></td><td>Used for peptide deprotection</td></tr><tr><td>N-Ethyldiisopropylamine</td><td>Alfa-Aesar (Lancashire, UK)</td><td></td><td>Used for tip modification</td></tr><tr><td>Triethylamine</td><td>Alfa-Aesar (Lancashire, UK)</td><td></td><td>Used for tip modification</td></tr><tr><td>Piperidine</td><td>Alfa-Aesar (Lancashire, UK)</td><td></td><td>Used for tip Fmoc deprotection</td></tr><tr><td>Fluorenylmethyloxycarbonyl-PEG-N-hydroxysuccinimide&amp;nbsp; (Fmoc-PEG-NHS)</td><td>Iris Biotech GmbH (Deutschland, Germany)</td><td></td><td>Used as the covalent flexible linker&amp;nbsp; (MW = 5,000 Da)</td></tr><tr><td>2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU)</td><td>Alfa Aser (Heysham, England)</td><td></td><td>Used as a coupling reagent.&amp;nbsp;</td></tr><tr><td>N-methyl-2-pyrrolidone (NMP)</td><td>Acros Organics (New Jersey, USA)</td><td></td><td>Used as Solvent in Tip modification procedure</td></tr><tr><td>DMF (dimethylformamide)</td><td>Merck (Darmstadt, Germany)</td><td></td><td>Used as Solvent in Tip modification procedure</td></tr><tr><td>Chloroform</td><td>Bio-Lab (Jerusalem, Israel)</td><td></td><td>Used as Solvent in Tip modification procedure</td></tr><tr><td>Ethanol (Anhydrous)</td><td>Gadot (Netanya, Israel)</td><td></td><td>Used as Solvent in Tip modification procedure</td></tr><tr><td>Trifluoro acetic acid (TFA)</td><td>Merck (Darmstadt, Germany)</td><td></td><td></td></tr><tr><td>Acetic anhydride</td><td>Merck (Darmstadt, Germany)</td><td></td><td></td></tr><tr><td>Peptides</td><td>GL Biochem (Shanghai, China).</td><td></td><td></td></tr><tr><td>Phenylalanine and Tyrosine&amp;nbsp;</td><td>Biochem (Darmstadt, Germany)&amp;nbsp;</td><td></td><td></td></tr><tr><td>30% TiO&lt;sub&gt;2&lt;/sub&gt; dispersion in the mixture of solvent 2-(2-Methoxyethoxy) ethanol (DEGME) and Ethyl 3-Ethoxypropionate (EEP)</td><td>Applied Vision Laboratories (Jerusalem, Israel)</td><td></td><td>(30%) in the mixture of solvent 2-(2 Methoxyethoxy) ethanol (DEGME) and Ethyl 3-Ethoxypropionate (EEP). Used for TiO&lt;sub&gt;2&lt;/sub&gt; substrate preparation</td></tr><tr><td>Mica substrates</td><td>TED PELLA, INC. (Redding, California, USA)</td><td></td><td>9.9 mm diameter</td></tr></tbody></table>

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.

Figure 1 exhibits the tip modification procedure. In the first step, a plasma treatment changes the surface of the silicon nitride tip. The tip presents OH groups. These groups will then react with the silanes. At the end of this step, the surface of the tip will be covered by free -NH2 groups. These free amines will then react with Fmoc -PEG-NHS, a covalent linker. The Fmoc group of the PEG linker is removed by pipyridine, a deprotecting reagent. Finally, the examined amino acid or peptide molecule is coupled through the free amine group of the PEG using the coupling reagent HBTU.
With the modified AFM tip it is possible to examine the interactions of the amino acid or peptide with the surface (Figure 2). The PEG molecule separates the peptide or amino acid from the tip and allows them to freely orient. A typical force measurement results in a Force-distance curve (Figure 3). This curve has a characteristic point of separation of the tip from the surface, and a single molecule adhesion event. The first peak indicates nonspecific interactions between the tip and the surface and the second peak refers to the specific adhesion event. From several hundred F-D curves it is possible to construct a histogram by plotting the number of adhesion events versus force. Applying a Gaussian fit on these histograms will determine the most probable force (MPF).
<img alt="Figure 1" src="/files/ftp_upload/54975/54975fig1.jpg" /> 
Figure 1:&#160;Tip modification procedure.&#160;Schematic representation of the chemical modification of the AFM tip. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54975/54975fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54975/54975fig2.jpg" /> 
Figure 2:&#160;SMFS experimental setup.&#160;Schematic illustration of the single-molecule force spectroscopy setup for measuring the interactions between amino acids or peptides and a desired surface. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54975/54975fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54975/54975fig3.jpg" /> 
Figure 3:&#160;Force-distance Curve.&#160;Typical single-molecule F-D curves for the rupture of (A) the peptide Gln-Pro-Ala-Ser-Ser-Arg-Tyr from a mica surface, and (B) the amino acid phenylalanine from a TiO2 surface. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54975/54975fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54975/54975fig4.jpg" /> 
Figure 4:&#160;The histograms plot the Most Probable Force (MPF) and the graphs plot the force Vs. loading rate.&#160;Typical histograms of the rupture force values of (A) the peptide Gln-Pro-Ala-Ser-Ser-Arg-Tyr from mica (at a loading rate of 3.1 &#177; 0.6 nN/s (N = 7 8)), (B) the amino acid phenylalanine from TiO2 (at loading rate of 4.2 &#177; 0.7 nN/s (N = 79)). The most probable force (MPF) value was calculated based on the Gaussian fit (the black lines). Loading-rate dependence for the rupture forces for (C) the peptide Gln-Pro-Ala-Ser-Ser-Arg-Tyr and (D) the amino acid phenylalanine. The kinetic parameters were extrapolated from the linear plot of the force vs. the logarithm of the apparent loading rate. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54975/54975fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy at JoVE.com

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

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 ...

insights-into-interactions-amino-acids-peptides-with-inorganic

Nanyang Technological University

Research

JoVE Journal

Chemistry

8.0K Views.  The Hebrew University of Jerusalem. 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.

Video: Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using 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. ...

Biochemistry

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 ...

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 ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer&#8217;s and Parkinson&#8217;s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

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.

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 ...

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 ...

Ensemble Force Spectroscopy by Shear Forces

Single-molecule techniques based on fluorescence and mechanochemical principles provide superior sensitivity in biological sensing. However, due to the lack of high throughput capabilities, the application of these techniques is limited in biophysics. Ensemble force spectroscopy (EFS) has demonstrated high throughput in the investigation of a massive set of molecular structures by converting mechanochemical studies of individual molecules into those of molecular ensembles. In this protocol, the DNA secondary structures (i-motifs) were unfolded in the shear flow between the rotor and stator of a homogenizer tip at shear rates up to 77796/s. The effects of flow rates and molecular sizes on the shear forces experienced by the i-motif were demonstrated. The EFS technique also revealed the binding affinity between DNA i-motifs and ligands. Furthermore, we have demonstrated a click chemistry reaction that can be actuated by shear force (i.e., mechano-click chemistry). These results establish the effectiveness of using shear force to control the conformation of molecular structures.

Ensemble force spectroscopy (EFS) is a robust technique for mechanical unfolding and real-time sensing of an ensemble set of biomolecular structures in biophysical and biosensing fields.

Single-molecule techniques based on fluorescence and mechanochemical principles provide superior sensitivity in biological sensing. However, due to the ...

A Fourier Transform Infrared Spectroscopy Technique to Study Peptide Self-Assembly

To prepare a self-assembly solution, dissolve 1 milligram of the lyophilized peptide powder in a mixture of 20% acetonitrile and 10-millimolar HEPES in a 1.5-milliliter microcentrifuge tube. Then, vortex the assembly solution and leave the sample to assemble at room temperature.
When the peptide assembly reaches maturation, dry 8 to 10 microliters of the assembly solution as a thin film on the attenuated total reflection diamond crystal, and monitor the disappearance of a large and broad water peak from 1,640 to 1,630 inverse centimeters as the dry film forms.
Acquire background and infrared spectra from 1,500 to 1,800 inverse centimeters, averaging 50 scans with a 2-inverse centimeter resolution, subtracting the background scans prior to each sample scan.
The infrared signature for beta-sheet assembly should be observed as a sharp peak between 1,625 and 1,635 inverse centimeters.