Analysis of DNA interactions using single-molecule force spectroscopy

Protein–DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein–DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide– and protein–DNA interactions are given.     

Protein interactions and misfolding analyzed by AFM force spectroscopy

Protein misfolding is conformational transition dramatically facilitating the assembly of protein molecules into aggregates of various morphologies. Spontaneous formation of specific aggregates, mostly amyloid fibrils, was initially believed to be limited to proteins involved in the development of amyloidoses. However, recent studies show that, depending on conditions, the majority of proteins undergo structural transitions leading to the appearance of amyloidogenic intermediates followed by aggregate formation. Various techniques have been used to characterize the protein misfolding facilitating the aggregation process, but no direct evidence as to how such a conformational transition increases the intermolecular interactions has been obtained as of yet. We have applied atomic force microscopy (AFM) to follow the interaction between protein molecules as a function of pH. These studies were performed for three unrelated and structurally distinctive proteins, alpha-synuclein, amyloid beta-peptide (Abeta) and lysozyme. It was shown that the attractive force between homologous protein molecules is minimal at physiological pH and increases dramatically at acidic pH. Moreover, the dependence of the pulling forces is sharp, suggesting a pH-dependent conformational transition within the protein. Parallel circular dichroism (CD) measurements performed for alpha-synuclein and Abeta revealed that the decrease in pH is accompanied by a sharp conformational transition from a random coil at neutral pH to the more ordered, predominantly beta-sheet, structure at low pH. Importantly, the pH ranges for these conformational transitions coincide with those of pulling forces changes detected by AFM. In addition, protein self-assembly into filamentous aggregates studied by AFM imaging was shown to be facilitated at pH values corresponding to the maximum of pulling forces. Overall, these results indicate that proteins at acidic pH undergo structural transition into conformations responsible for the dramatic increase in interprotein interaction and promoting the formation of protein aggregates.     

Atomic force microscopy-based force spectroscopy--biological and biomedical applications

The use of atomic force microscopy (AFM) applied to biological systems to generate high resolution images is gaining a wider acceptance. However, the most remarkable advances are being achieved on the use of the AFM to measure inter- and intramolecular interaction forces with piconewton resolution, not only to demonstrate this ability but also actually to solve biological and biomedical relevant questions. Single-molecule force spectroscopy recognition studies enable the detection of specific interaction forces, based on the AFM sensitivity and the possibility of manipulating individual molecules. In this review, we describe the basic principles of this methodology and some of the practical aspects involved. The ability to measure interactions at the single-molecule level is illustrated by some relevant examples. A special focus is given to the study of the fibrinogen-erythrocyte binding and its relevance as a cardiovascular risk factor. An approach to the latter problem by single-molecule force spectroscopy allowed the molecular recognition, characterization, and partial identification of a previously unknown receptor for fibrinogen on human erythrocytes.     

Detecting molecular interactions that stabilize, activate and guide ligand-binding of the sodium/proton antiporter MjNhaP1 from Methanococcus jannaschii

Integral membrane proteins are involved in virtually every cellular process. Precisely regulating these machineries would allow controlling many human and vertebrate diseases. Embedded into cellular membranes, membrane proteins establish molecular interactions that sensitively react to environmental changes and to molecular compounds, such as ligands or inhibitors. We applied atomic force microscopy (AFM) to image the Na(+)/H(+) antiporter MjNhaP1 from Methanococcus jannaschii, and single-molecule force spectroscopy (SMFS) to probe molecular interactions that drive the protein structure-function relationship. High-resolution AFM topographs showed the dimeric assembly of MjNhaP1 being reconstituted into a lipid bilayer. SMFS of MjNhaP1 unraveled molecular interactions stabilizing individual structural domains. Transmembrane domains exhibited certain probabilities to unfold individually or cooperatively with other domains resulting in different unfolding pathways. Helices VIII and X established pH sensitive interactions altering significantly upon MjNhaP1 activation, while removal of the ligand (Na(+)) destabilized the entire antiporter except helix VIII. It is assumed that Asp234/235 of helix VIII are involved in the ligand-binding site and that helix X plays a functional role in the activation of the transporter.     

Synthesis and characterization of biocompatible thermo-responsive gelators based on ABA triblock copolymers

The synthesis of biocompatible, thermo-responsive ABA triblock copolymers in which the outer A blocks comprise poly(N-isopropylacrylamide) and the central B block is poly(2-methacryloyloxyethyl phosphorylcholine) is achieved using atom transfer radical polymerization with a commercially available bifunctional initiator. These novel triblock copolymers are water-soluble in dilute aqueous solution at 20 degrees C and pH 7.4 but form free-standing physical gels at 37 degrees C due to hydrophobic interactions between the poly(N-isopropylacrylamide) blocks. This gelation is reversible, and the gels are believed to contain nanosized micellar domains; this suggests possible applications in drug delivery and tissue engineering.     

Single molecular dynamic interactions between glycophorin A and lectin as probed by atomic force microscopy

Glycophorin A (GpA) is one of the most abundant transmembrane proteins in human erythrocytes and its interaction with lectins has been studied as model systems for erythrocyte related biological processes. We performed a force measurement study using the force mode of atomic force microscopy (AFM) to investigate the single molecular level biophysical mechanisms involved in GpA-lectin interactions. GpA was mounted on a mica surface or natively presented on the erythrocyte membrane and probed with an AFM tip coated with the monomeric but multivalent Psathyrella velutina lectin (PVL) through covalent crosslinkers. A dynamic force spectroscopy study revealed similar interaction properties in both cases, with the unbinding force centering around 60 pN with a weak loading rate dependence. Hence we identified the presence of one energy barrier in the unbinding process. Force profile analysis showed that more than 70% of GpAs are free of cytoskeletal associations in agreement with previous reports.     

Automated force volume image processing for biological samples

Atomic force microscopy (AFM) has now become a powerful technique for investigating on a molecular level, surface forces, nanomechanical properties of deformable particles, biomolecular interactions, kinetics, and dynamic processes. This paper specifically focuses on the analysis of AFM force curves collected on biological systems, in particular, bacteria. The goal is to provide fully automated tools to achieve theoretical interpretation of force curves on the basis of adequate, available physical models. In this respect, we propose two algorithms, one for the processing of approach force curves and another for the quantitative analysis of retraction force curves. In the former, electrostatic interactions prior to contact between AFM probe and bacterium are accounted for and mechanical interactions operating after contact are described in terms of Hertz-Hooke formalism. Retraction force curves are analyzed on the basis of the Freely Jointed Chain model. For both algorithms, the quantitative reconstruction of force curves is based on the robust detection of critical points (jumps, changes of slope or changes of curvature) which mark the transitions between the various relevant interactions taking place between the AFM tip and the studied sample during approach and retraction. Once the key regions of separation distance and indentation are detected, the physical parameters describing the relevant interactions operating in these regions are extracted making use of regression procedure for fitting experiments to theory. The flexibility, accuracy and strength of the algorithms are illustrated with the processing of two force-volume images, which collect a large set of approach and retraction curves measured on a single biological surface. For each force-volume image, several maps are generated, representing the spatial distribution of the searched physical parameters as estimated for each pixel of the force-volume image.     

AFM for structure and dynamics of biomembranes

We review structure and dynamic measurements of biomembranes by atomic force microscopy (AFM). We focus mainly on studies involving supported lipid bilayers (SLBs), particularly formation by vesicle rupture on flat and corrugated surfaces, nucleation and growth of domains in phase-separated systems, anesthetic-lipid interactions, and protein/peptide interactions in multicomponent systems. We show that carefully designed experiments along with real-time AFM imaging with superior lateral and z resolution (0.1 nm) have revealed quantitative details of the mechanisms and factors controlling vesicle rupture, domain shape and size, phase transformations, and some model biological interactions. The AFM tip can also be used as a mechanical transducer and incorporated in electrochemical measurements of membrane components; therefore, we touch on these important applications in both model and cell membranes.     

AFM for structure and dynamics of biomembranes

We review structure and dynamic measurements of biomembranes by atomic force microscopy (AFM). We focus mainly on studies involving supported lipid bilayers (SLBs), particularly formation by vesicle rupture on flat and corrugated surfaces, nucleation and growth of domains in phase-separated systems, anesthetic-lipid interactions, and protein/peptide interactions in multicomponent systems. We show that carefully designed experiments along with real-time AFM imaging with superior lateral and z resolution (0.1 nm) have revealed quantitative details of the mechanisms and factors controlling vesicle rupture, domain shape and size, phase transformations, and some model biological interactions. The AFM tip can also be used as a mechanical transducer and incorporated in electrochemical measurements of membrane components; therefore, we touch on these important applications in both model and cell membranes.     

Synthesis of biocompatible,stimuli-responsive,physical gels based on ABA triblock copolymers

ABA triblock copolymers [A = 2-(diisopropylamino)ethyl methacrylate), DPA or 2-(diethylamino)ethyl methacrylate), DEA; B = 2-methacryloyloxyethyl phosphorylcholine, MPC] prepared using atom transfer radical polymerization dissolve in acidic solution but form biocompatible free-standing gels at around neutral pH in moderately concentrated aqueous solution (above approximately 10 w/v % copolymer). Proton NMR studies indicate that physical gelation occurs because the deprotonated outer DPA (or DEA) blocks become hydrophobic, which leads to attractive interactions between the chains: addition of acid leads to immediate dissolution of the micellar gel. Release studies using dipyridamole as a model hydrophobic drug indicate that sustained release profiles can be obtained from these gels under physiologically relevant conditions. More concentrated DPA-MPC-DPA gels give slower release profiles, as expected. At lower pH, fast, triggered release can also be achieved, because gel dissolution occurs under these conditions. Furthermore, the nature of the outer block also plays a role; the more hydrophobic DPA-MPC-DPA triblock gels are formed at lower copolymer concentrations and retain the drug longer than the DEA-MPC-DEA triblock gels.     

Correlated fluorescence quenching and topographic mapping of Light-Harvesting Complex II within surface-assembled aggregates and lipid bilayers

Light-Harvesting Complex II (LHCII) is a chlorophyll-protein antenna complex that efficiently absorbs solar energy and transfers electronic excited states to photosystems I and II. Under excess light intensity LHCII can adopt a photoprotective state in which excitation energy is safely dissipated as heat, a process known as Non-Photochemical Quenching (NPQ). In vivo NPQ is triggered by combinatorial factors including transmembrane ΔpH, PsbS protein and LHCII-bound zeaxanthin, leading to dramatically shortened LHCII fluorescence lifetimes. In vitro, LHCII in detergent solution or in proteoliposomes can reversibly adopt an NPQ-like state, via manipulation of detergent/protein ratio, lipid/protein ratio, pH or pressure. Previous spectroscopic investigations revealed changes in exciton dynamics and protein conformation that accompany quenching, however, LHCII-LHCII interactions have not been extensively studied. Here, we correlated fluorescence lifetime imaging microscopy (FLIM) and atomic force microscopy (AFM) of trimeric LHCII adsorbed to mica substrates and manipulated the environment to cause varying degrees of quenching. AFM showed that LHCII self-assembled onto mica forming 2D-aggregates (25–150?nm width). FLIM determined that LHCII in these aggregates were in a quenched state, with much lower fluorescence lifetimes (~0.25?ns) compared to free LHCII in solution (2.2–3.9?ns). LHCII-LHCII interactions were disrupted by thylakoid lipids or phospholipids, leading to intermediate fluorescent lifetimes (0.6–0.9?ns). To our knowledge, this is the first in vitro correlation of nanoscale membrane imaging with LHCII quenching. Our findings suggest that lipids could play a key role in modulating the extent of LHCII-LHCII interactions within the thylakoid membrane and so the propensity for NPQ activation.     

An atomic force microscopy study of Galleria mellonella apolipophorin III effect on bacteria

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. As revealed by LIVE/DEAD staining, incubation of Gram-negative and Gram-positive bacteria in the presence of Galleria mellonella apoLp-III led to growth inhibition of selected bacteria. An atomic force microscopy (AFM) study of bacterial cells after apoLp-III treatment showed considerable alterations in the cell surface of Bacillus circulans, Klebsiella pneumoniae and Salmonella typhimurium. Our results clearly demonstrate that apoLp-III disturbed the proper structure of the bacterial cell surface. The alterations were dissimilar to those caused by cationic antimicrobial peptide, cecropin B, suggesting a different mode of action against bacteria. The present results indicate that AFM provides a powerful tool for studying the interactions of apoLp-III with microbial cells.     

AFM studies of the supramolecular assembly of bacterial photosynthetic core-complexes

The atomic force microscope (AFM) allows visualization of the assembly and molecular interactions of single proteins. Most recently, AFM images of bacterial membranes have revealed details of the supramolecular architecture of bacterial photosynthetic apparatus in different species. The near-native experimental conditions used in AFM imaging reduce artefacts and make AFM ideal for studying native conformations. High-resolution AFM of native membranes has revealed variation in core-complex architectures amongst species.     

Influence of nonuniform geometry on nanoindentation of viral capsids

A series of recent nanoindentation experiments on the protein shells (capsids) of viruses has established atomic force microscopy (AFM) as a useful framework for probing the mechanics of large protein assemblies. Specifically these experiments provide an opportunity to study the coupling of the global assembly response to local conformational changes. AFM experiments on cowpea chlorotic mottle virus, known to undergo a pH-controlled swelling conformational change, have revealed a pH-dependent mechanical response. Previous theoretical studies have shown that homogeneous changes in shell geometry can play a significant role in the mechanical response. This article develops a method for accurately capturing the heterogeneous geometry of a viral capsid and explores its effect on mechanical response with a nonlinear continuum elasticity model. Models of both native and swollen cowpea chlorotic mottle virus capsids are generated from x-ray crystal structures, and are used in finite element simulations of AFM indentation along two-, three-, and fivefold icosahedral symmetry orientations. The force response of the swollen capsid model is observed to be softer by roughly a factor of two, significantly more nonlinear, and more orientation-dependent than that of a native capsid with equivalent elastic moduli, demonstrating that capsid geometric heterogeneity can have significant effects on the global structural response.     

Multidimensional atomic force microscopy for drug discovery: A versatile tool for defining targets,designing therapeutics and monitoring their efficacy

Current therapeutic design involves combinatorial chemistry and system biology-based molecular synthesis and bulk pharmacological assays. Therapeutics delivery is usually non-specific to disease targets and requires excessive dosage. Efficient therapeutic discovery and delivery would require molecular level understanding of the therapeutics–effectors (e.g., channels and receptors) interactions and their cell and tissue responses. This review summarizes the application of multidimensional scanning probe techniques, especially atomic force microscopy (AFM), for drug discovery. Important features of AFM include its capability of atomic scale structural and physical properties study of live biological systems, its open architecture that allows its integration with other techniques, tools and operating environments, and its application for creating and characterizing nanocarriers and implantable vehicles for controlled delivery. Specific areas covered include: 1) the operating principle and examples of AFM integrated with electrical recording, fluorescence imaging and microfluidics, (2) examples of AFM nanoscale imaging that has provided new paradigms in pathogenesis, including protein misfolding diseases (e.g., Alzheimer's disease, cancer, diabetes) and diseases arising from environmental and life choices and thus identifies potential therapeutic targets, (3) high-throughput parallel sensors, comprising integrated cantilevered microarrays, TIRF, microfluidics and nanoelectronics, for potential rapid diagnosis of pathogens, allergens and biomarkers as well as for therapeutics design, (4) the definition target macromolecules and structures, using intermolecular interaction assays, (5) the definition of abnormal vs normal tissues and the assessment of therapeutic efficacy by monitoring biomechanics, and (6) the development and characterization of nanocarrier-based drug delivery (e.g., nanoliposomes and nanoparticles) systems that allow high efficiency in vivo or the topical administration of a small dosage of therapeutics.     

The impact of fluoxetine and pH values on relaxation of the ternary lipid monolayers

Fluoxetine (FLX), used in the clinic to treat depression, is a well-known cationic amphiphilic antidepressant. To get a deeper insight into the effect of FLX on Langmuir monolayers, in this study the stability and relaxation of 1,2-dioctadecanoyl-sn-glycero-3-phophocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol (DSPC/POPC/CHOL) monolayers without and with FLX at different pH values were studied. The experiments involved surface pressure-area (π-A) measurements, mean molecular area-time (A-t) measurements, and atomic force microscope (AFM) analysis. It was found that intermolecular interactions decreased after the addition of FLX in the subphase but increased with increasing pH values. The relaxation of the ternary lipid monolayers with FLX was dominated by dissolution steps, and the dissolution rates decreased with increasing pH values. These findings can be easily confirmed by the analysis of thermodynamic parameters calculated for the investigated films. The data obtained in this study help to understand the effect of drugs on the ternary lipid monolayers from the molecular point of view.     

On protein partitioning in two-phase aqueous polymer systems.

The partitioning of proteins between the coexisting phases of two-phase aqueous polymer systems reflects an intricate and delicate balance of interactions between proteins, polymers, salts and water. Experimental investigations have suggested that a large number of factors influence protein partitioning, including the types of polymers, their molecular weight and concentration; the protein sizes, conformation and composition; salt type and concentration, and solution pH; and the presence of ligands attached to the polymer which may interact with surface sites of the protein. Complementary modelling attempts have been successful in illuminating several molecular-level mechanisms influencing protein partitioning using lattice-model techniques, viral expansions and a scaling-thermodynamic approach. In spite of these experimental and modelling approaches, many of the physical phenomena associated with these complex systems are not well understood. Notably, the precise nature of the protein-polymer interactions and the potent effect of inorganic salts on the partitioning of proteins in these systems remains poorly understood.     

Seawater at the nanoscale: marine gel imaged by atomic force microscopy

The present study introduces atomic force microscopy (AFM) as a tool for characterization of marine gel network and marine biopolymers self-assembly, not accessible by other techniques. AFM imaging of marine gel samples collected in summers 2003 and 2004 in the northern Adriatic Sea provided insight into molecular organization of gel network and associations between polysaccharide fibrils in the network. Initial stages of biopolymers self-assembly were visualized by AFM in a phytoplankton bloom experiment performed in the same aquatorium. Based on AFM imaging and differential scanning calorimetry, the marine gel is characterized as a thermoreversible physical gel and the dominant mode of gelation as crosslinking of polysaccharide fibrils by hydrogen bonding which results in helical structures and their associations. Direct deposition of whole seawater on freshly cleaved mica followed by rinsing was the procedure that caused the least impact on the original structures of biopolymer assemblies in seawater.     

Measurement of interaction force between nanoarrayed integrin alphavbeta3 and immobilized vitronectin on the cantilever tip

Protein nanoarrays containing integrin alphavbeta3 or BSA were fabricated on ProLinker-coated Au surface by dip-pen nanolithography (DPN). An atomic force microscope (AFM) tip coated with ProLinker was modified by vitronectin. We measured the interaction force between nanoarrayed integrin alphavbeta3 or BSA and immobilized vitronectin on the cantilever tip by employing tethering-unbinding method. The unbinding force between integrin alphavbeta3 and vitronectin (1087+/-62 pN) was much higher than that of between BSA and vitronectin (643+/-74 pN). These results demonstrate that one can distinguish a specific protein interaction from non-specific interactions by means of force measurement on the molecular interactions between the nanoarrayed protein and its interacting protein on the AFM tip.