Characterization of Recognition Events between Proteins on a Single

Review pubs.acs.org/IECR

Characterization of Recognition Events between Proteins on a Single Molecule Level with Atomic Force Microscopy Yang Xie,† Jianhua Wang,*,† and Yonglai Feng‡ †

Key Laboratory of Biorheological Science and Technology, Ministry of Education College of Bioengineering, Chongqing University, Chongqing, 400044, P. R. China ‡ Exposure and Biomonitoring Division, Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario K1A 0K9, Canada ABSTRACT: The application of functional imaging tools and techniques on the molecular level enables investigators to characterize structures of biomolecules and interactions between biomolecules. The characterization of recognition events between biomolecules with atomic force microscopy (AFM) is a new methodology in biological research. Recent advances in the field of scanning probe microscopy especially atomic force microscopy have made it possible to discover the dynamic systems of molecular self-assembly and quantify the molecular forces between proteins. The interaction mechanisms between biomolecules were explored by many researchers to tackle broad-category initiatives of human diseases. This review overviews the advances in characterizing recognition events between proteins on the molecular level, specifically on antigen−antibody interactions and the aggregation of amyloid-β peptides. AFM as an emerging approach for characterizing biomolecular interactions will also be highlighted. Furthermore, the potential and limitations of AFM for the measurement of single molecule interactions and the issues in analysis procedure will be discussed.

1. INTRODUCTION AFM has become a trustworthy instrument which contributed a lot to researches in the biochemical field. In AFM studies, topographic imaging presents the detail of sample surface morphology and force spectroscopy provides the detail of the interaction force between molecules.1,2 It was reported that the interactions between proteins such as antigen−antibody interactions3,4 and misfolding of amyloid-β proteins5 could result in human diseases. Therefore, probing and characterizing the molecular interactions of the relevant functional proteins hold great significance in the development of new pharmaceutical and clinical medicines. Furthermore, study on the interactions between proteins on the single molecule level is fundamentally important to researches in life science. Recently, AFM as a new scanning probe microscopic technology has been widely applied to the analysis of biological surfaces for properties of adhesive force and frictional force between biomolecules, such as proteins.6 The AFM enables us to nanoprobe the molecular structure of protein-modified surfaces. Compared to the STM technique that is limited by metal graininess, AFM can achieve native information on dynamic molecular events, including information on the binding of receptor−ligand pairs, molecular motors, protein folding pathways, and the DNA-binding agents.7 Pastre and his colleagues reported a method to measure the accessibility of DNA adsorbance to restriction endonucleases (EcoRI and EcoRV) using an AFM system.8 The AFM technique now is evolving from a qualitative imaging tool to a quantitative dynamic tool that can determine mechanical properties of biomolecules or living cells.9 The relevant data on recognition events between single molecules from AFM technology had been widely applied to research and development for molecular biology.11−13 A momentous advantage in AFM technique is to © 2016 American Chemical Society

study the interactions between biomolecules on the single molecule level. The distinct in situ imaging capabilities and high force sensibility enables AFM to be a promising tool to achieve real-time information on the development of quaternary structure and self-assembly. Advances in probe design and the application of single molecule force spectroscopy, for example, the meliorated repertoire of imaging mode, including contact or tapping mode are considered as integral parts of the AFM technique. The protein structure is stabilized by the incorporation of linkers, which provides a uniform surface and limits the number of possible orientations. For example, the IgG protein molecules are immobilized on gold surface through the ordered and stable mercapto self-assembled monolayer with covalent bond.24 By “feeling” the surface of a protein sample with a tiny probe tip, AFM can capture highresolution images with lateral resolution of approximately 0.5 nm and vertical resolution of approximately 0.1 nm. The application of AFM opened new perspectives in the investigation of biological surfaces either in air or in liquid. Cantilevers with different resonance frequency and spring constant are employed in different AFM modes. Generally, lowresonance frequency and soft cantilevers are more applicable to image samples in contact and tapping mode in liquid. Nevertheless, high-resonance frequency and stiff cantilevers are more suitable for tapping mode in air. In contact mode, the probe tip is in contact with the investigated surface during scanning, causing damage to the specimens, especially on soft specimens like living cells. Molecules are often pushed away by Received: Revised: Accepted: Published: 1469

October 22, 2015 January 1, 2016 January 26, 2016 January 26, 2016 DOI: 10.1021/acs.iecr.5b03922 Ind. Eng. Chem. Res. 2016, 55, 1469−1476

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Industrial & Engineering Chemistry Research

Figure 1. Experimental setup of SMFS (a). One of the interacting Aβ42 molecules is immobilized on the APS modified mica surface via a long PEG linker. The counterpart Aβ42 molecule is anchored on the MAS functionalized AFM tip. A typical approach-retraction cycle of recorded rupture force curve (b). The rupture events and the polymer stretching segment of the force curve are indicated with a single-headed arrow and a double headed arrow, respectively.

interactions between peptides that dynamically change their properties via physicochemical mechanisms in this review.

the probe tip in contact mode. To overcome the disadvantage of contact mode, tapping mode of AFM has been developed.10 In tapping mode, the stylus oscillates touch the sample only at the end of its downward movement. Thus, the contact time and the friction forces are reduced. Nevertheless, tapping mode lacked the resolution acquired by contact mode. The AFM technique has been increasingly applied to the measurement of physical properties such as viscoelastic properties for biological molecules. Recently, an experimental method was developed for the determination of the unbinding force between two single biomolecules and phosphate buffered saline (PBS) with a combination of self-assembled monolayer (SAM) for sample preparation and atomic force microscopy (AFM) for measurement of specific interaction forces between proteins.11 The recognition of interactions between proteins was sensitive under a variety of environmental stimuli, including physicochemical (pH, temperature, and electric field) and biochemical (presence of specific molecules) inputs.12 da Silva and co-workers exploited specific interactions between enzyme acetolactate synthase (ALS) and ALS-inhibitor herbicides by functionalizing the AFM tips with ALS and modifying the substrate with the herbicides.13 Actually, the molecular recognition of antibodies to membrane-antigens and the extraction of the antigens from membranes on the single molecule level were investigated by Kienberger’s group in as early as 2005.14 However, there are no damages in the biological function of proteins detected in these papers. Due to its applicability on the visualization of biological samples in liquid phase, AFM was proposed as a new approach to characterize recognition force between proteins in various circumstance conditions and select effective drug molecule.15 Although this approach can address pharmacological questions on the molecular level, the application of AFM on interactions between biomolecules on the single molecule level still has great potential to bring a new method for modern biomedicine treatments. This review will introduce recent advances in characterizing mechanical behaviors between proteins on the molecular level, specifically, antigen−antibody interactions and misfolding of amyloid β protein (Aβ). We will also discuss the

2. THE AFM TECHNIQUE As a meaningful functional and structural parameter of biomolecules, intermolecular forces (such as adhesion force and friction force) have been investigated for characterizing the recognition events in the native environments by single molecule spectroscopy. AFM is nondestructive in characterizing specific recognition events between biomolecules in air and liquid. In the past, AFM was usually used to investigate the forces within a single biomolecule or between biomolecules.16,17 The applications of atomic force microscopy can be divided into two aspects: morphology and mechanics. Atomic force microscopy was first applied to obtain molecular dynamic images of the sample surface. Afterward, AFM was increasingly used for quantitative analysis of the molecular mechanics forces. It was once considered impossible to measure the adhesion or friction forces between molecules with the sensitivity about 1 pN. In AFM, the rate of imaging and resolution of images depend on sensitivity of vibrating motion of AFM piezoelectric microcantilever. Resonance frequency and oscillation amplitudes of cantilever are of utmost importance in AFM application. The high resolution of forces results from the small spring constants of the cantilever (usually in the range of 0.5−0.01 N/m).1 With aid of AFM technique, it is feasible to record unbinding forces at the single molecule level by measuring the rupture force between a ligand and a receptor molecule. The dynamic information on molecules can be obtained through changing the loading rate.18 The specific and nonspecific interaction forces between proteins are therefore quantified. The dynamic change process of protein structure can also be monitored by AFM. In AFM, one interacting partner is immobilized on a substrate and the other binding partner is connected to a probe tip. The AFM tip is then contacted to the substrate. When the tip is pulled away from the surface of protein monolayer, the binding of receptor−ligand pair is broken and the rupture force will be measured. In the way of single molecule force spectroscopy (SMFS), AFM can measure rupture force 1470

DOI: 10.1021/acs.iecr.5b03922 Ind. Eng. Chem. Res. 2016, 55, 1469−1476

Review

Industrial & Engineering Chemistry Research between the biological molecules binding to the substrate and to the probe tip respectively so as to characterize the molecular interaction (Figure 1).40 Development on the nanoprobing technique in AFM on the single molecular level has provided new insights into the molecular mechanism for specific recognition events between biological molecules. To date, AFM has been widely applied to characterizing biological molecular recognition processes because of its high force sensitivity and the capability of operating under different physiological conditions. When the AFM tip is contacting with the surface, a protein− ligand complex is formed. With the ligand-functionalized tip being retracted from the surface of protein until the bond breaks, the rupture force (i.e., unbinding force) will be recorded and force−distance curves will be output. The force curve can provide important information on the study of recognition events between proteins from a theoretical view. Force− distance curves have been employed for characterization of some important mechanical behaviors between proteins. Several types of measurement, including determination of adhesion force, surface charge densities, and degrees of hydrophobicity were used to quantify the behaviors.19,20 By measuring the adhesion force between the tip of an atomic force microscope cantilever modified with antigen and substrate attached to the antibody, the different interactions between proteins under the condition of different drug molecules in physiological solution can be determined for selection of eutherapeutic and less toxic drug molecules for pharmacological use.21 The natural immobilization of proteins is an appropriate technique for the single molecule force spectroscopic studies,22 as an efficient sample stabilization processing method that is essential for the scanning of interactions between proteins by the probe. Authors achieved some important findings through depositing soluble proteins on layers, through direct physical adsorption or chemical bonds between molecule and substrate. Ebner and his colleagues used a new cross-linker that has two different amino-reactive functions, of which one end is a carboxyl (N-hydroxysuccinimide ester) group and the other end is a benzaldehyde group. The former can react with the amino groups of the tips much faster than the latter, although the reactivity of the latter is sufficient to covalently bind lysine residues of proteins via Schiff base formation. The method has been well validated using biotinylated IgG as a bioligand on the tip and mica-bound avidin as the complementary receptor (Figure 2).23 In the study of the biological relevant protein molecule, one of the crucial disadvantages in the application of AFM is the sample pretreatment. The protein sample needs to be treated carefully prior to AFM analysis, otherwise the protein molecules might be damaged or dragged away by the probe. Therefore, it is important to develop a fast, accurate, and noninvasive sample pretreatment method in the study of interactions between proteins. This limitation is obvious particularly in the liquid phase environment, in which the probe motion is enough to induce the solubilization effects of protein molecules. The binding between the protein monolayer and the substrate must be strong enough to protect the sample from being dragged away by the probe, and meanwhile, there should be no structural variations appearing because of the movement of probe tip. Some other factors may also result in these variations, such as the structure collapse induced by the surface change during drying or freezing.

Figure 2. Schematic representation of the testing system for tip− antibody linking via lysine−aldehyde coupling. In the first step (in organic solvent), the NHS−ester of aldehyde−PEG−NHS reacts with the amino groups on the tip, while in the second step (in aqueous buffer), one of the antibody’s lysines forms a Schiff base with the aldehyde function on the free end of the cross-linker. The Schiff base is reduced by NaCNBH3 to form an irreversible bond (not shown). When brought into contact with avidin-covered mica, the biotin residues on the model antibody show reversible binding to, and unbinding from, immobilized avidin.

3. EXPERIMENTAL RESEARCH PROGRESS This review will be restricted to experimentally observed recognition events between biological proteins with the aforementioned AFM technique. The knowledge of mechanical behaviors between proteins is critical to understanding the f pharmacological action and designing a better medicine. Atomic force microscopy provides a novel approach to directly measure individual protein−protein forces under near-physiological conditions. A protein was linked onto the surface of the AFM tip, and force−distance curves were measured on the substrate modified by a self-assembled monolayer (SAM), allowing the current AFM experiments to be mostly performed in solution environment in vitro.24 The biorecognition process of protein−protein pairs has been studied more and more from both experimental and theoretical points of view. The pairs contain various categories of associations with different properties. There are two kinds of typical protein pairs in the experimental studies. One pair is the antigen−antibody complexes that are usually characterized by tight binding, long lifetime, and high specificity. Another one is the short-lived transient complexes that are formed by molecules such as protein folding. In the past decade, studies on single molecule force studies helped researchers to design experiments to characterize specific recognition events between biological proteins. Kim and colleagues examined the interaction between the proteins attached to the tip and lipid bilayer biomembranes including CD14 in both the presence and the absence of the antimicrobial peptide, polymyxin B (PMB) using the method 1471


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event that is affected by forces acting at the nanoscale. Information on the interaction force between single biomolecules allows a comprehensive understanding of the interaction mechanisms. An antibody, for example, immunoglobulins, is the major soluble effector molecule produced by an immune cell under the presence of an exogenous molecule, an antigen. The noncovalent interactions for formation of the antigen−antibody complex include hydrogen and ionic bindings and hydrophobic and van der Waals interactions. The noncovalent bindings are weaker interactions compared to the covalent bindings. Clearly, the specific and nonspecific interactions of antigen with antibody, initially at the membrane of B lymphocytes and subsequently in circulation, play an effective defensive role for the antibody response. As a soluble CD4 (sCD4), anti-CD4 antibody and antigp120 antibody are considered as entry inhibitors in the human immunodeficiency virus (HIV) therapy. Chen and co-workers investigated nonbinding interaction forces between sCD4 and gp120, CD4 antigen and antibody, as well as gp120 and antigen−antibody as important information on mechanical behaviors to understand the effects of viral entry inhibitors on HIV infection.33 They found that the nonbinding interaction forces between sCD4 and gp120, CD4 antigen and antibody, and gp120 antigen and antibody were 25.45 ± 20.46 pN, 51.22 ± 34.64 pN, and 89.87 ± 44.63 pN, respectively. In their study, the interaction force for gp120-sCD4 was much larger than the interaction force between gp120 and cell-surface CD4 (∼26 pN) as was the same for the CD4 or gp120 antigen−antibody interactions. This may partially elucidate the mechanical reason why HIV entry inhibitors (such as sCD4, anti-CD4 antibody, anti-gp120antibody, and others) can inhibit HIV infection. Recently, Wakayama and co-workers investigated the effect of temperature on the dissociation process of the complex of βlactoglobulin and antibovine β-lactoglobulin IgG polyclonal antibody using AFM.34 In this work, the nonbinding interaction forces between β-lactoglobulin and the antibody were measured at temperatures of 25, 35, and 45 °C. The results indicated that the nonbinding interaction forces decreased when temperature increased (Figures 4 and 5). Interestingly, the nonbinding interaction force exhibited two linear regimes at each temperature (Figure 5). Similarly, Jauvert and co-workers investigated single molecule force interactions between glutathione-S-transferase (GST) and its cognate antibody immobilized on dendritips.35 3.2. Misfolding of Protein. The β-amyloid fibrilization has been studied as a pathological related protein with relative success. Neurodegenerative diseases are raising more attentions in the world because they are fatal and there is no treatment available. Therefore, providing symptom treatment for the aging population is becoming not only a medical but also a financial challenge. The accumulation of amyloid β-peptide (Aβ) in the brain is a characterization of Alzheimer disease. Currently, physiological mechanisms on the aggregation behavior and factors of Aβ are still not clearly understood. Several recent researches indicated that metal ions might produce an effect on Aβ aggregation and fibril formation. In 2011, Kim and colleagues applied AFM to quantitatively characterize interactions of Aβ40 peptides over a broad range of pH values.36 With a similar approach, Yu and colleagues evaluated influences of several compounds on the protein aggregation process. The results demonstrated that Zn2+ or Al3+ cations dramatically increased α-synuclein interactions in unfavorable conditions for α-synuclein misfolding (neutral

of self-assembled monolayer, and atomic force microscopy. Their results showed that the antimicrobial peptide affected the molecular interaction between lipopolysaccharide (LPS) and immune proteins.25 Successively, Okada and his co-workers employed this method to evaluate the binding affinity of peptide probes for profilin (protein). Each peptide probe was immobilized on a cantilever tip and a mica substrate, respectively. The interaction force to profiling was examined by force curve measurements. Their results on the retraction forces revealed a sequence-dependent affinity of the peptide probe for profilin.26 Ikuta and co-workers investigated the interaction between a ligand and its receptor at the single molecule level on the surface of living cells. The results revealed a difference between the interactions of diferric transferrin (Tf) with TfR2α and the interactions of Tf with TfR1. This difference may be attributed to the different physiological roles of the two receptors.27 With a decrease of correspondingly iron uptake rates, the study achieved the association constant of 5.6 × 106 M−1 for the binding of Tf to TfR2α, about 50 times lower than the binding of Tf to TfR1. Moreover, the recognition events between ligand−receptor pairs have been investigated for understanding the physiological roles of these biological proteins at molecular level. These pairs include an anticancer peptide fragment of azurin (a bacterial protein that can be internalized in cancer cells and induce apoptosis) with p53,28 p53 with Mdm2,29 ribonuclease barnase with its inhibitor barstar,30 and actin filament and binding protein.31 Bonazza and co-workers also utilized AFM to investigate the complex formation between the von Willebrand factor (VWF) and factor VIII (FVIII), two essential hemostatic components of human blood.32 Their results indicated that the fraction of VWF knots bound to FVIII was 25 ± 4%, and declined to 2 ± 1% when a complex was formed in the presence of the high salt buffer containing 500 mM Ca2+ (Figure 3). Their results demonstrated that the reaction of such molecules with binding partners could be monitored by imaging them on a solid substrate. 3.1. Antigen−Antibody Partner. The formation of a complex between antigen−antibody partners is a recognition

Figure 3. Summary of the single-molecule approach results. Shown is the fraction of VWF globular units that exhibited changes due to attachment of FVIII molecules under different experimental conditions. The percentages in this diagram have been determined by directly imaging identical VWF molecules before and after exposure to FVIII on the mica surface. Binding of FVIII to VWF was observed in PBS buffer, whereas it was almost totally inhibited in a high ionic strength buffer. Blank denotes a control experiment performed with PBS buffer in the absence of FVIII. 1472


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played the key role.39 Next, Hane and co-workers measured the rupture force for dissociating two Aβ(1−42) peptides in the presence of Cu2+ using atomic force spectroscopy, and the aggregation of Aβ was resolved.40 The result of this work suggested that the reduction of lag time associated with Aβ aggregation on a single molecule level resulted from increasing binding forces at the initial stage of interactions between two Aβ peptides. Recently, Lv and co-workers41 applied single molecule AFM force spectroscopy to elucidate the role of Cu2+ cations on interactions of interpeptides through the immobilization of one of two interacting Aβ42 molecules on a mica surface while tethering the counterpart molecule onto the tip. This work showed that substoichiometric Cu2+ cations accelerated the formation of fibrils at pH 7.4 (Figure 6) and 5.0, whereas no effect of Cu2+ cations was observed at pH 4.0. Taken together, the combination of AFM force spectroscopy and imaging analyses demonstrated that Cu2+ cations promoted both the initial and the elongation stages of Aβ aggregation, and protein protonation diminished the effect of Cu2+ on the other hand.

Figure 4. Representative distributions of the unbinding forces between anti-β-lactoglobulin antibody immobilized on the cantilever and βlactoglobulin immobilized on the mica measured at different loading rates and at different temperatures. (A) Distribution of forces measured at 25 °C. (B) Distribution of forces measured at 45 °C. All histograms were normalized to the total count of detection events that originated from the specific interaction between anti-βlactoglobulin antibody and β-lactoglobulin. The solid lines at each histogram correspond to double-Gaussian function fittings used to extract the most probable unbinding force. Figure reproduced from ref 34. Copyright 2012 American Chemical Society.

4. DATA ANALYSIS AND THEORETICAL MODEL Besides visually observing the difference between the AFM topography of a protein monolayer or a cell surface,42,43 much work is needed on the data analysis of molecular rupture events. Force−distance or approach−retract curves, which present the motion of the piezoelectric tube scanner on the zaxis, are obtained by bringing the ligand-modified tip in contact with the receptor-immobilized substrate. When a ligand interacts with a receptor, a mutation part (defined as rupture force or unbinding force) in the withdrawing stage of the curve will be observed. Until now, there has beeb no universal method to finish the statistical analysis of AFM data for the single molecule systems.44−46 The unbinding force between the characterized molecule pair is usually influenced by the parameters of degree of molecular freedom, molecular orientation, and affinity. In actual AFM studies, one of the major difficulties of data analysis is the presence of multiple bonds. Therefore, the Bell−Evans’ model is applied to analyze the ligand−receptor bond rupture events in most of the single molecule studies. Methods varying from one to another have been proposed to select rupture events from data points of the observed retract curve at various loading rates.47,48 A large number of observed retract curves obtained at various loading rates are gathered before making a Bell-Evans plot and estimating the most probable rupture forces. Usually, experimental data are fitted with Gaussian distribution (Figure 4) to obtain the average rupture force for ligand−receptor binding.34 However, the number of selected bins has a major impact on the shape of the probability distribution histogram. Theoretically, finer bins seem more befitting than larger ones, although a large number of data are required. Several years ago, Filomena used the Bell, Evans, and Ritchie model to extract kinetic bond rupture parameters.49 In this paper, the bondspecific parameters: koff (the unstressed dissociation rate), 1/koff (the bond lifetime), and xu (the width of the potential barrier) were calculated from the dependence of the rupture forces on the loading rate. By predicting a linear correlation between the most probable rupture force (F*) and the logarithm of the most probable loading rate (LR*), it is possible to build the Bell−Evans plot (Figure 7). From a Bell−Evans model, the energy barrier height (related to dissociation constant koff) can be obtained by

Figure 5. Dynamic force spectra (most probable unbinding force versus loading rate) for the anti-β-lactoglobulin antibody-β-lactoglobulin interaction. The first peaks in the distribution of the unbinding force measured at each loading rate and temperature were used as the most probable force (see Data Analysis of the Measurements of the Temperature Effect on Parameters of the Kinetics and Energy Landscape by AFM in the Experimental Procedures section of ref 34). Figure reproduced from ref 34. Copyright 2012 American Chemical Society.

pH).37 Subsequently, Shimanouchi and co-workers first observed the growth behavior of Aβ fibrils in the presence of liposomes and metal ion (copper(II) ion). The influences of those additives on the growth behavior of amyloid fibrils were also quantitatively investigated to estimate the inhibitory effect.38 The result of this work indicated that copper(II) ion showed the strong inhibitory effect on the growth of Aβ fibrils in a bulk aqueous solution. Successively, Lovas and co-workers combined AFM and Molecular Dynamics (MD) simulations for characterizing the misfolding process of an Aβ peptide. The authors found that misfolding of the Aβ peptide proceeded via the loss of conformational flexibility and formation of stable dimers, suggesting that the subsequent Aβ aggregation process 1473


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Figure 6. AFM force spectroscopy in the presence and absence of Cu2+ cations at pH 7.4. The force spectroscopy in the absence of Cu2+ cations is shown in the upper panel. The columns include, from left to right: the overlap of all raw force curves, the distribution of contour length, and the distribution of rupture force. The lower panel shows the corresponding characteristic of force spectroscopy in the presence of Cu2+ cations. The Lc and Fr denote the most probable contour length and the most probable rupture force, respectively. Figure reproduced with permission from ref 41. Copyright 2012 Springer.

extrapolating the fitting line until F* = 0 and the energy barrier width (xβ) can be extrapolated from the slope of the fitting line, respectively. The information on the energy landscape using energy barriers described in the Bell−Evans model provides useful detail of the binding chemistry. For example, in Teulon’s paper, the width of an energy barrier measured at 1 Å or less is defined to indicate that the rupture of hydrogen bonds or salt bridges may exist.50 In addition, some scientific groups have studied the reaction kinetics of ligand−receptor interactions. For instance, Zhang and co-workers proposed a method to reveal the detailed relationships between the real-time CBM3a concentration on a cellulose surface, reaction time, and initial CBM3a concentration in solution.51

The specific and nonspecific unbinding events could be distinguished reliably, and the structure and functionality of the molecular partners should be preserved appropriately. Although many efforts have been made to gradually consummate the immobilization method, a definite theory to standardize the experimental strategy and the data analysis is still lacking. Above all, the most appropriate strategy for the molecular self-assembly should be determined according to the corresponding requirement of the experimental system under analysis. Because of the single molecule detection sensitivity and the thimbleful of sample demand in reliable experiments, the AFM technique may have the remarkable potentiality to connect with the biorecognition-based theory for the development of early pharmacology. Combining the real-time topographical imaging and characterization of the binding details of single molecular partners is expected to become a meaningful approach for researches to extract simultaneous spatial and dynamical information on the interacting partners on the single molecule level. Furthermore, the association of AFM with ultrasensitive detectors, such as advanced fluorescence, should lead to an innovative approach for the study of biorecognition events between proteins. Finally, from the view of clinical practice, investigating the biologically relevant interactions between proteins using atomic force spectroscopy will provide much valuable knowledge that may potentially serve as an important parameter in the treatment of diseases. Indeed, the single molecule spectroscopy based methods are distinct from those traditional NMR and UV−vis based methods. The former provides macro information on molecular recognition in the experimental system. The advantage of AFM on carrying out studies under physiological conditions, with the possibility to change environmental factors such as pH, enables AFM to be a tool

5. CONCLUSION AND PROSPECTIVE Atomic force microscopy (AFM) has taken a prominent position in terms of single molecular mechanics research for its ability to investigate molecular interactions. AFM can measure rupture forces between single molecular pairs immobilized on the tip and the substrate in physiological solution, respectively, at the pico-Newton level, which elucidates the interaction behaviors of biomolecules on a single molecule level. This holds a remarkable significance by offering dynamic details of biomolecules, ligands, and drugs during the biorecognition process. The results reported in a large number of literature have further promoted the development of theoretical models to reveal the mechanisms of the recognition events between biomolecules. While the unbinding force trend as a function of the logarithm of the loading rate can be achieved through the Bell−Evans model, the binding free energy can be evaluated through the Jarzynski theoretical model. A crucial preparation prior to AFM imaging is to immobilize the biomolecules on the surfaces of substrate and tip. However, the recognition tests consist of only two molecular partners. 1474



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REFERENCES

(1) Zlatanova, J.; Lindsay, S. M.; Leuba, S. H. Single molecule force spectroscopy in biology using the atomic force microscope. Prog. Biophys. Mol. Biol. 2000, 74, 37−61. (2) Rief, M.; Grubmueller, H. Force spectroscopy of single biomolecules. ChemPhysChem 2002, 3, 255−261. (3) Kienberger, F.; Kada, G.; Mueller, H.; Hinterdorfer, P. Single molecule studies of antibody-antigen interaction strength versus intramolecular antigen stability. J. Mol. Biol. 2005, 347, 597−606. (4) Li, M.; Xiao, X. B.; Liu, L. Q.; Xi, N.; Wang, Y. C.; Dong, Z. L.; Zhang, W. J. Atomic force microscopy study of the antigen-antibody binding force on patient cancer cells based on ROR1 fluorescence recognition. J. Mol. Recognit. 2013, 26, 432−438. (5) Karran, E.; Mercken, M.; De Strooper, B. The amyloid cascade hypothesis for Alzheimer’s disease: an appraisal for the development of therapeutics. Nat. Rev. Drug Discovery 2011, 10, 698−712. (6) Kim, I. H.; Lee, H. Y.; Lee, H. D.; Jung, Y. J.; Tendler, S. J. B.; Williams, P. M.; Allen, S.; Ryu, S. H.; Park, J. W. Interactions between signal-transducing proteins measured by atomic force microscopy. Anal. Chem. 2009, 81, 3276−3284. (7) Gross, L.; Mohn, F.; Moll, N.; Meyer, G.; Ebel, R.; AbdelMageed, W. M.; Jaspars, M. Organic structure determination using atomic-resolution scanning probe microscopy. Nat. Chem. 2010, 2, 821−825. (8) Pastre, D.; Hamon, L.; Sorel, I.; Le Cam, E.; Curmi, P. A.; Pietrement, O. Specific DNA−Protein interactions on mica investigated by atomic force microscopy. Langmuir 2010, 26, 2618−2623. (9) Muller, S. A.; Muller, D. J.; Engel, A. Assessing the structure and function of single biomolecules with scanning transmission electron and atomic force microscopes. Micron 2011, 42, 186−195. (10) Hansma, P. K.; Cleveland, J. P.; Radmacher, M.; Walters, D. A.; Hillner, P. E.; Bezanilla, M.; Fritz, M.; Vie, D.; Hansma, H. G.; Prater, C. B.; Massie, J.; Fukunaga, L.; Gurley, J.; Elings, V. Tapping mode atomic force microscopy in liquids. Appl. Phys. Lett. 1994, 64, 1738− 1740. (11) Lv, Z. J.; Wang, J. H.; Chen, G. P.; Deng, L. H. Probing specific interaction forces between human IgG and rat anti-human IgG by selfassembled monolayer and atomic force microscopy. Nanoscale Res. Lett. 2010, 5, 1032−1038. (12) Bizzarri, A. R.; Cannistraro, S. Antigen−antibody biorecognition events as discriminated by noise analysis of force spectroscopy curves. Nanotechnology 2014, 25, 335102. (13) da Silva, A. C. N.; Deda, D. K.; Bueno, C. C.; Moraes, A. S.; Da Roz, A. L.; Yamaji, F. M.; Prado, R. A.; Viviani, V.; Oliveira, O. N.; Leite, F. L. Nanobiosensors exploiting specific interactions between an enzyme and herbicides in atomic force spectroscopy. J. Nanosci. Nanotechnol. 2014, 14, 6678−6684. (14) Kienberger, F.; Kada, G.; Mueller, H.; Hinterdorfer, P. Single molecule studies of antibody-antigen interaction strength versus intramolecular antigen stability. J. Mol. Biol. 2005, 347, 597−606. (15) Zhang, J.; Wu, G. M.; Song, C. L.; Li, Y. J.; Qiao, H. Y.; Zhu, P.; Hinterdorfer, P.; Zhang, B. L.; Tang, J. L. Single molecular recognition force spectroscopy study of a luteinizing hormone-releasing hormone analogue as a carcinoma target drug. J. Phys. Chem. B 2012, 116, 13331−13337. (16) Hinterdorfer, P.; Dufrene, Y. F. Detection and localization of single molecular recognition events using atomic force microscopy. Nat. Methods 2006, 3, 347−55. (17) Dupres, V.; Menozzi, F. D.; Locht, C.; Clare, B. H.; Abbott, N. L.; Cuenot, S.; Bompard, C.; Raze, D.; Dufrêne, Y. F. Nanoscale mapping and functional analysis of individual adhesions on living bacteria. Nat. Methods 2005, 2, 515−20. (18) Drew, M. E.; Chworos, A.; Oroudjev, E.; Hansma, H.; Yamakoshi, Y. A tripod molecular tip for single molecule ligandreceptor force spectroscopy by AFM. Langmuir 2010, 26, 7117−25. (19) Dufrene, Y. F.; Martinez-Martin, D.; Medalsy, I.; Alsteens, D.; Muller, D. J. Multiparametric imaging of biological systems by forcedistance curve−based AFM. Nat. Methods 2013, 10, 847−852.

Figure 7. Theoretical Bell−Evans plot. The upper graph indicated how the Bell−Evans plot is built from the most probable rupture forces (F*). These forces are obtained from a Gaussian fit of the force distribution obtained at a given effective loading rate. The lower graph indicates how to extract the energy barrier properties: the width (xβ in nm) and the kinetic dissociation rate (koff in s−1). The width xβ is obtained from the slope: xβ = kBT/slope. The koff value is obtained by extrapolating the loading rate value at F* = 0: koff = rF=0xβ/kBT.

for probing of dynamic events in biomolecules especially proteins. It has been demonstrated that AFM is able to measure the binding force between proteins in different drug solutions. Accordingly, specific and nonspecific (physicochemical) interactions between proteins can be calculated from force curves. From the viewpoint of new drug exploitation, it is meaningful to study the interactions between small molecules and diseaserelated proteins, which may answer biological questions of human diseases.

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AUTHOR INFORMATION

Corresponding Author

*Tel.:0086-23-65102507. Fax: 0086-2365103031. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Foundation and Advanced Research Project of CQ CSTC (2013jjB0011), and Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province. 1475


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Characterization of Recognition Events between Proteins on a Single

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