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Block experiment. (a and b) Topography and recognition images of UCP1 molecules in the lipid membrane acquired with an ATP-tethered tip, prior to blocking. (c and d) Topography and recognition images after blocking by addition of free ATP into the solution while scanning the same position. Almost all recognition spots (black) disappeared, demonstrating the specificity of the UCP1-ATP interaction.
Source publication
We combined recognition imaging and force spectroscopy to study the interactions between receptors and ligands on the single molecule level. This method allowed the selection of a single receptor molecule reconstituted in a supported lipid membrane at low density, with the subsequent quantification of the receptor-ligand unbinding force. Based on a...
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... specificity of the recognition signals was proven by a block experiment. Here ~8.6 mM of free ATP was injected into the buffer solution and effectively blocked the binding sites for ATP in the UCP1 binding pocket (Figs. 5(b, d)). The nearly complete loss of the recognition spots after addition of free ATP (Figs. 5(b, d)), while leaving the topography image unchanged (Figs. 5(a, c)), clearly confirms that most of the recognition spots were caused by specific binding of tip-linked ATP to UCP1 molecules in the planar lipid membrane. This coincides with already published results for the UCP1-ATP interaction.35, ...
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... specificity of the recognition signals was proven by a block experiment. Here ~8.6 mM of free ATP was injected into the buffer solution and effectively blocked the binding sites for ATP in the UCP1 binding pocket (Figs. 5(b, d)). The nearly complete loss of the recognition spots after addition of free ATP (Figs. 5(b, d)), while leaving the topography image unchanged (Figs. 5(a, c)), clearly confirms that most of the recognition spots were caused by specific binding of tip-linked ATP to UCP1 molecules in the planar lipid membrane. This coincides with already published results for the UCP1-ATP interaction.35, ...
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... specificity of the recognition signals was proven by a block experiment. Here ~8.6 mM of free ATP was injected into the buffer solution and effectively blocked the binding sites for ATP in the UCP1 binding pocket (Figs. 5(b, d)). The nearly complete loss of the recognition spots after addition of free ATP (Figs. 5(b, d)), while leaving the topography image unchanged (Figs. 5(a, c)), clearly confirms that most of the recognition spots were caused by specific binding of tip-linked ATP to UCP1 molecules in the planar lipid membrane. This coincides with already published results for the UCP1-ATP interaction.35, ...
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... specificity of the recognition signals was proven by a block experiment. Here ~8.6 mM of free ATP was injected into the buffer solution and effectively blocked the binding sites for ATP in the UCP1 binding pocket (Figs. 5(b, d)). The nearly complete loss of the recognition spots after addition of free ATP (Figs. 5(b, d)), while leaving the topography image unchanged (Figs. 5(a, c)), clearly confirms that most of the recognition spots were caused by specific binding of tip-linked ATP to UCP1 molecules in the planar lipid membrane. This coincides with already published results for the UCP1-ATP interaction.35, ...
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... specificity of the recognition signals was proven by a block experiment. Here ~8.6 mM of free ATP was injected into the buffer solution and effectively blocked the binding sites for ATP in the UCP1 binding pocket (Figs. 5(b, d)). The nearly complete loss of the recognition spots after addition of free ATP (Figs. 5(b, d)), while leaving the topography image unchanged (Figs. 5(a, c)), clearly confirms that most of the recognition spots were caused by specific binding of tip-linked ATP to UCP1 molecules in the planar lipid membrane. This coincides with already published results for the UCP1-ATP interaction.35, ...
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... specificity of the recognition signals was proven by a block experiment. Here ~8.6 mM of free ATP was injected into the buffer solution and effectively blocked the binding sites for ATP in the UCP1 binding pocket (Figs. 5(b, d)). The nearly complete loss of the recognition spots after addition of free ATP (Figs. 5(b, d)), while leaving the topography image unchanged (Figs. 5(a, c)), clearly confirms that most of the recognition spots were caused by specific binding of tip-linked ATP to UCP1 molecules in the planar lipid membrane. This coincides with already published results for the UCP1-ATP interaction.35, ...
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... specificity of the recognition signals was proven by a block experiment. Here ~8.6 mM of free ATP was injected into the buffer solution and effectively blocked the binding sites for ATP in the UCP1 binding pocket (Figs. 5(b, d)). The nearly complete loss of the recognition spots after addition of free ATP (Figs. 5(b, d)), while leaving the topography image unchanged (Figs. 5(a, c)), clearly confirms that most of the recognition spots were caused by specific binding of tip-linked ATP to UCP1 molecules in the planar lipid membrane. This coincides with already ...
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... specificity of the recognition signals was proven by a block experiment. Here ~8.6 mM of free ATP was injected into the buffer solution and effectively blocked the binding sites for ATP in the UCP1 binding pocket (Figs. 5(b, d)). The nearly complete loss of the recognition spots after addition of free ATP (Figs. 5(b, d)), while leaving the topography image unchanged (Figs. 5(a, c)), clearly confirms that most of the recognition spots were caused by specific binding of tip-linked ATP to UCP1 molecules in the planar lipid membrane. This coincides with already published results for the UCP1-ATP interaction.35, ...
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... signals was proven by a block experiment. Here ~8.6 mM of free ATP was injected into the buffer solution and effectively blocked the binding sites for ATP in the UCP1 binding pocket (Figs. 5(b, d)). The nearly complete loss of the recognition spots after addition of free ATP (Figs. 5(b, d)), while leaving the topography image unchanged (Figs. 5(a, c)), clearly confirms that most of the recognition spots were caused by specific binding of tip-linked ATP to UCP1 molecules in the planar lipid membrane. This coincides with already published results for the UCP1-ATP interaction.35, ...
Citations
... TREC uses a dynamic mode and feedback loop-based electronics that relates the reduction of the oscillation amplitude with specific interactions between a receptor on the sample and a ligand attached to the AFM tip. Numerous biological systems have been interrogated by TREC, such as avidin: biotin [217], human ether-à-go-go related (hERG) K + channels:antibody Kv 11.1 [218], human clusterin:anti-clusterin [219], lipid bilayers [220], among others. TDFM operation consists of oscillating the functionalized AFM tip with tuning forks which are made of quartz materials. ...
Biomolecular interactions underpin most processes inside the cell. Hence, a precise and quantitative understanding of molecular association and dissociation events is crucial, not only from a fundamental perspective, but also for the rational design of biomolecular platforms for state-of-the-art biomedical and industrial applications. In this context, atomic force microscopy (AFM) appears as an invaluable experimental technique, allowing the measurement of the mechanical strength of biomolecular complexes to provide a quantitative characterization of their interaction properties from a single molecule perspective. In the present review, the most recent methodological advances in this field are presented with special focus on bioconjugation, immobilization and AFM tip functionalization, dynamic force spectroscopy measurements, molecular recognition imaging and theoretical modeling. We expect this work to significantly aid in grasping the principles of AFM-based force spectroscopy (AFM-FS) technique and provide the necessary tools to acquaint the type of data that can be achieved from this type of experiments. Furthermore, a critical assessment is done with other nanotechnology techniques to better visualize the future prospects of AFM-FS.
... For some negatively charged biomolecules (e.g., DNA), divalent cations such as Ni 21 and Mg 21 are added to the solution to bridge the negative charges of the biomolecule and the substrate. (2) Covalent immobilization methods: The biomolecule of interest can be attached to silicon and silicon nitride tip/substrate via silanization chemistry (Koehler et al., 2017) or to a gold tip/substrate via goldÀthiol chemistry (Martines et al., 2012). ...
... Biomolecule-inserted artificially supported lipid bilayers can also be obtained for AFM studies. Biomolecules can be inserted into artificially supported lipid bilayers by the proteoliposome fusion method (Koehler et al., 2017) or the reconstituted liposome fusion method (Abdulreda, Bhalla, Chapman, & Moy, 2008). ...
Polysaccharide is a natural polymer that has good gelation, emulsifying, and rheological properties, and has a wide application in the food industry. Meanwhile, its structural properties have a close relationship to its functional behaviors. As a nanotechnology, atomic force microscopy (AFM) could be applied to describe the structural properties of polysaccharides. In this chapter, the morphology, structures, and their changes, as well as the gelling properties of polysaccharides, were revealed using AFM. What’s more, food component mixtures could induce novel properties of food systems for their new structures. Therefore AFM could be used to study the intermolecular interactions in complex systems and investigate the real structure of complexes of natural materials. These will benefit the understanding of the intra and intermolecular interaction forces of polysaccharides and other food components.
... AFM consists of a probe (flexible cantilever with a sharp tip at the end), piezoelectric scanner, cantilever deflection detecting system or a spilt photodiode, AFM electronics and feedback loop, and cantilever and sample holders (shown in Figure 1B). The characterization of any substrate by AFM (Koehler et al., 2017;Li et al., 2017) is done by measuring the force of interaction between the cantilever tip and sample surface (obtained via measurement of cantilever deflection). The probe is first approached toward the substrate manually, and this is followed by a fine motion of the cantilever tip, controlled by the piezoelectric scanner, and it depends on the force set point chosen by the user. ...
Since its invention, atomic force microscopy (AFM) has come forth as a powerful member of the “scanning probe microscopy” (SPM) family and an unparallel platform for high-resolution imaging and characterization for inorganic and organic samples, especially biomolecules, biosensors, proteins, DNA, and live cells. AFM characterizes any sample by measuring interaction force between the AFM cantilever tip (the probe) and the sample surface, and it is advantageous over other SPM and electron micron microscopy techniques as it can visualize and characterize samples in liquid, ambient air, and vacuum. Therefore, it permits visualization of three-dimensional surface profiles of biological specimens in the near-physiological environment without sacrificing their native structures and functions and without using laborious sample preparation protocols such as freeze-drying, staining, metal coating, staining, or labeling. Biosensors are devices comprising a biological or biologically extracted material (assimilated in a physicochemical transducer) that are utilized to yield electronic signal proportional to the specific analyte concentration. These devices utilize particular biochemical reactions moderated by isolated tissues, enzymes, organelles, and immune system for detecting chemical compounds via thermal, optical, or electrical signals. Other than performing high-resolution imaging and nanomechanical characterization (e.g., determining Young’s modulus, adhesion, and deformation) of biosensors, AFM cantilever (with a ligand functionalized tip) can be transformed into a biosensor (microcantilever-based biosensors) to probe interactions with a particular receptors of choice on live cells at a single-molecule level (using AFM-based single-molecule force spectroscopy techniques) and determine interaction forces and binding kinetics of ligand receptor interactions. Targeted drug delivery systems or vehicles composed of nanoparticles are crucial in novel therapeutics. These systems leverage the idea of targeted delivery of the drug to the desired locations to reduce side effects. AFM is becoming an extremely useful tool in figuring out the topographical and nanomechanical properties of these nanoparticles and other drug delivery carriers. AFM also helps determine binding probabilities and interaction forces of these drug delivery carriers with the targeted receptors and choose the better agent for drug delivery vehicle by introducing competitive binding. In this review, we summarize contributions made by us and other researchers so far that showcase AFM as biosensors, to characterize other sensors, to improve drug delivery approaches, and to discuss future possibilities.
... What else lies ahead? Other possibilities offered by AFM force spectroscopy remain to be tapped into; for example, dynamic force spectroscopy can be applied to quantify out-of-equilibrium thermodynamic and kinetic parameters of single-molecular interactions as has been demonstrated for single virus-cell interactions by the Alsteens group [71][72][73][74][75][76][77][78]. This methodology can serve as an excellent platform to investigate the effect of small-molecule inhibitors of adhesive interactions under physiologically relevant conditions. ...
It is an understatement that mating and DNA transfer are key events for living organisms. Among the traits needed to facilitate mating, cell adhesion between gametes is a universal requirement. Thus, there should be specific properties for the adhesion proteins involved in mating. Biochemical and biophysical studies have revealed structural information about mating adhesins, as well as their specificities and affinities, leading to some ideas about these specialized adhesion proteins. Recently, single-cell force spectroscopy (SCFS) has added important findings. In SCFS, mating cells are brought into contact in an atomic force microscope (AFM), and the adhesive forces are monitored through the course of mating. The results have shown some remarkable characteristics of mating adhesins and add knowledge about the design and evolution of mating adhesins.
... Faster localization of binding sites can be achieved with topography and recognition imaging 107,108 , in which tips functionalized with ligands are driven at resonance frequencies to simultaneously acquire topography and recognition images on cells 109 . Although this mode does not directly quantify interaction forces, it is equipped with the capability to conduct force spectroscopy experiments at binding sites localized in recognition images 110 . ...
... Correlative fluorescence force spectroscopy imaging 56,228,230,237,[240][241][242] Correlated topography, adhesion and elasticity 49,[51][52][53][54]69,107,141 Receptor density and clustering 49,109,110,[212][213][214][215][216] Chemical imaging 72,[84][85][86] AFM-FS, atomic force microscopy-based force spectroscopy. ...
Physical forces and mechanical properties have critical roles in cellular function, physiology and disease. Over the past decade, atomic force microscopy (AFM) techniques have enabled substantial advances in our understanding of the tight relationship between force, mechanics and function in living cells and contributed to the growth of mechanobiology. In this Primer, we provide a comprehensive overview of the use of AFM-based force spectroscopy (AFM-FS) to study the strength and dynamics of cell adhesion from the cellular to the single-molecule level, spatially map cell surface receptors and quantify how cells dynamically regulate their mechanical and adhesive properties. We first introduce the importance of force and mechanics in cell biology and the general principles of AFM-FS methods. We describe procedures for sample and AFM probe preparations, the various AFM-FS modalities currently available and their respective advantages and limitations. We also provide details and recommendations for best usage practices, and discuss data analysis, statistics and reproducibility. We then exemplify the potential of AFM-FS in cellular and molecular biology with a series of recent successful applications focusing on viruses, bacteria, yeasts and mammalian cells. Finally, we speculate on the grand challenges in the area for the next decade. Atomic force microscopy-based force spectroscopy can probe the strength and dynamics of cell adhesion to understand how physical forces influence cellular function, physiology and disease. Here, Dufrêne and colleagues discuss the ability of this technology to work as an ultra-sensitive force sensor to study the adhesion and elasticity of complex biological systems including viruses, bacteria, yeasts and mammalian cells.
... After the completement of the tip functionalization and materials interface, now it is ready to perform SMFS through approaching and retracting the AFM tip onto and from the materials surface. The operation modes of the SMFS experiments could be classified into dynamic force spectroscopy (DFS) [45][46][47], force mapping (FM) [48,49], and force clamping (FC) [50,51]. ...
The quantification of the interactions between biomolecules and materials interfaces is crucial for design and synthesis functional hybrid bionanomaterials for materials science, nanotechnology, biosensor, biomedicine, tissue engineering, and other applications. Atomic force spectroscopy (AFM)-based single-molecule force spectroscopy (SMFS) provides a direct way for measuring the binding and unbinding forces between various biomolecules (such as DNA, protein, peptide, antibody, antigen, and others) and different materials interfaces. Therefore, in this review, we summarize the advance of SMFS technique for studying the interactions between biomolecules and materials interfaces. To achieve this aim, firstly we introduce the methods for the functionalization of AFM tip and the preparation of functional materials interfaces, as well as typical operation modes of SMFS including dynamic force spectroscopy, force mapping, and force clamping. Then, typical cases of SMFS for studying the interactions of various biomolecules with materials interfaces are presented in detail. In addition, potential applications of the SMFS-based determination of the biomolecule-materials interactions for biosensors, DNA based mis-match, and calculation of binding free energies are also demonstrated and discussed. We believe this work will provide preliminary but important information for readers to understand the principles of SMFS experiments, and at the same time, inspire the utilization of SMFS technique for studying the intermolecular, intramolecular, and molecule-material interactions, which will be valuable to promote the reasonable design of biomolecule-based hybrid nanomaterials.
... Simultaneous acquisition of topographical and qualitative ligand binding information has been achieved with the invention of the AFM-based topography and recognition imaging (TREC), which has been extensively used to map the organization and binding of several ligand-receptor pairs, such as the human gonadotropin-releasing hormone receptor and the distribution of Hsp70 on the surface of cancer cells [18]. Although TREC does not allow the quantitative extraction of binding parameters from the ligand-receptor recognition, it can be coupled to SMFS to retrieve the quantitative parameters of binding on selected receptors [19]. ...
Cell surface receptors, often called transmembrane receptors, are key cellular components as they control and mediate cell communication and signalling, converting extracellular signals into intracellular signals. Elucidating the molecular details of ligand binding (cytokine, growth factors, hormones, pathogens,...) to cell surface receptors and how this binding triggers conformational changes that initiate intracellular signalling is needed to improve our understanding of cellular processes and for rational drug design. Unfortunately, the molecular complexity and high hydrophobicity of membrane proteins significantly hamper their structural and functional characterization in conditions mimicking their native environment. With its piconewton force sensitivity and (sub)nanometer spatial resolution, together with the capability of operating in liquid environment and at physiological temperature, atomic force microscopy (AFM) has proven to be one of the most powerful tools to image and quantify receptor-ligand bonds in situ under physiologically relevant conditions. In this article, a brief overview of the rapid evolution of AFM towards quantitative biological mapping will be given, followed by selected examples highlighting the main advances that AFM-based ligand-receptor studies have brought to the fields of cell biology, immunology, microbiology, and virology, along with future prospects and challenges. Graphical abstract Graphical abstract
... Recently, we compared binding forces between different nucleotides and UCP1-UCP3 at the single molecule level using a combination of recognition imaging and force spectroscopy (Koehler et al., 2017). We revealed that bond lifetimes of both mUCP3-PN and mUCP1-PN interactions decreased in proportion to the degree of PN phosphorylation. ...
... For the first time, the combination of recognition and force modes of atomic force microscopy (AFM) (Koehler et al., 2017) have allowed estimation of the depth of the nucleotide binding side from the membrane surface. It was shown to be 1.27 nm (Zhu et al., 2013). ...
Membrane uncoupling protein 3 (UCP3), a member of the mitochondrial uncoupling protein family, was discovered in 1997. UCP3′s properties, such as its high homology to other mitochondrial carriers, especially to UCP2, its short lifetime and low specificity
of UCP3 antibodies, have hindered progress in understanding its biological function and transport mechanism over decades. The abundance of UCP3 is highest in murine brown adipose tissue (BAT, 15.0 pmol/mg protein), compared to heart (2.7 pmol/mg protein) and the gastrocnemius muscle (1.7 pmol/mg protein), but it is still 400-fold lower than the abundance of UCP1, a biomarker for BAT. Investigation of UCP3 reconstituted in planar bilayer membranes revealed that it transports protons only when activated by fatty acids (FA). Although purine nucleotides (PN) inhibit UCP3-mediated transport, the molecular mechanism differs from that of UCP1. It remains a conundrum that two homologous proton-transporting proteins exist within the same tissue. Recently, we proposed that UCP3 abundance directly correlates with the degree of FA β-oxidation in cell metabolism. Further development in this field implies that UCP3 may have dual function in transporting substrates, which have yet to be identified, alongside protons. Evaluation of the literature with respect to UCP3 is a complex task because (i) UCP3 features are often extrapolated from its “twin” UCP2 without additional proof, and (ii) the specificity of antibodies against UCP3 used in studies is rarely evaluated. In this review, we primarily focus on recent findings obtained for UCP3 in biological and biomimetic systems.
(PDF) Important Trends in UCP3 Investigation. Available from: https://www.researchgate.net/publication/332765546_Important_Trends_in_UCP3_Investigation [accessed May 06 2019].
... Since UCP can only be reconstituted in the supported lipid membrane at low lateral density, a combination of recognition imaging and force spectroscopy for mapping and studying the interaction between mUCP1 and PNs was applied as described in Koehler et al. [28]. Proteins that showed strong binding in the TREC mode were selected for further detailed analysis using force spectroscopy. ...
... To elucidate whether PN-UCP bond lifetime determines PN inhibitory potency, we applied a combination of topographical, recognition (TREC) and force modes of AFM [28]. Attachment of PN to the cantilever enabled the identification and quantification of protein-nucleotide interactions simultaneously to target protein imaging and provided information about UCP-topography and UCP-PN binding (Fig. 3, A, inset). ...
... In 2017, a combination of two techniques, simultaneous topographical and recognition (TREC) imaging and SMFS was suggested by Koehler et al. [7]. TREC and SMFS are two AFM techniques that ideally complement each other. ...
... Topography and recognition images of reconstituted receptors are recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor molecules are then selected from the recognition image with nanometer resolution before the AFM is switched to the force spectroscopy mode, using positional feedback control [7]. The combined mode is an optimal tool for dynamic force probing on different pre-selected molecules, resulting in higher throughput when compared with force mapping. ...
... The proof-of-concept was shown for the quantitative characterization of the binding mechanism between mitochondrial membrane uncoupling protein 1 (UCP1) and its inhibitor adenosine triphosphate (ATP). Here the binding dynamics as well as the interaction energy landscape was elucidated [7,14] (Section 4.1). In the second example, the interaction between the bacterial translocation channel SecYEG and the cytoplasmic motor-protein SecA was characterized. ...
Ligand binding to receptors is one of the most important regulatory elements in biology as it is the initiating step in signaling pathways and cascades. Thus, precisely localizing binding sites and measuring interaction forces between cognate receptor–ligand pairs leads to new insights into the molecular recognition involved in these processes. Here we present a detailed protocol about applying a technique, which combines atomic force microscopy (AFM)-based recognition imaging and force spectroscopy for studying the interaction between (membrane) receptors and ligands on the single molecule level. This method allows for the selection of a single receptor molecule reconstituted into a supported lipid membrane at low density, with the subsequent quantification of the receptor–ligand unbinding force. Based on AFM tapping mode, a cantilever tip carrying a ligand molecule is oscillated across a membrane. Topography and recognition images of reconstituted receptors are recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor molecules are selected from the recognition image with nanometer resolution before the AFM is switched to the force spectroscopy mode, using positional feedback control. The combined mode allows for dynamic force probing on different pre-selected molecules. This strategy results in higher throughput when compared with force mapping. Applied to two different receptor–ligand pairs, we validated the presented new mode.
