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.     

Direct force measurements of the streptavidin-biotin interaction

The interaction between streptavidin and its ligand, biotin, were studied by direct force measurements. The complimentary approaches of surface force apparatus (SFA) and atomic force microscopy (AFM) were used to elucidate both long-range and short-range adhesive interactions of the streptavidin biotin interaction. The high spatial resolution of the SFA provided a detailed profile of the intersurface forces of apposing surfaces functionalized with streptavidin and biotin. Measurements obtained by the SFA corresponded to long and intermediate-range forces that are important in determining ligand receptor association. AFM was used to measure the unbinding force of individual streptavidin biotin complexes. These measurements revealed the short-range interactions (i.e. hydrophobic and hydrogen bonding forces) that stabilize the intermolecular bond.     

On the temperature dependence of complex formation between chitosan and proteins

Chitosan is a biocompatible easily degradable polysaccharide, which, because of its positive charge, is able to interact favorably with deprotonated carboxyl groups of proteins. The strength of these charge-charge interactions is generally low, resulting in poor colloidal stability of the complexes. To investigate if other noncovalent forces contribute to stabilizing such systems, we have selected α-lactalbumin, β-lactoglobulin, β-casein, and human growth hormone, characterized by a common acidic pI value (～ 5) that ensures their overall negative charge at physiological pH. Binding energetics between chitosan and proteins was studied by isothermal titration calorimetry, whereas the thermal stability was assessed by differential scanning calorimetry. Our data show that colloidal stability of the particles depends on protein identity as well as temperature, indicating the involvement of nonelectrostatic interactions (e.g., hydrophobic effect) as driving forces for the complex formation. This suggests that chitosan-protein drug delivery systems can be improved through preparation process optimization with regard to temperature.     

Chemical force microscopy of cellulosic fibers

Atomic force microscopy with chemically modified cantilever tips (chemical force microscopy) was used to study the pull-off forces (adhesion forces) on cellulose model surfaces and bleached softwood kraft pulp fibers in aqueous media. It was found that for the –COOH terminated tips, the adhesion forces are dependent on pH, whereas for the –CH₃ and –OH terminated tips adhesion is not strongly affected by pH. Comparison between the cellulose model surfaces and cellulosic fibers under our experimental conditions reveal that surface roughness does not affect adhesion strongly. X-ray photoelectron spectroscopy (XPS) and Fourier Transformed Infrared (FTIR) spectroscopy reveal that both substrate surfaces have homogeneous chemical composition. The results show that chemical force microscopy can be used for the chemical characterization of cellulose surfaces at a nano-level.     

Direct force measurements between cellulose surfaces and colloidal silica particles

The interaction of cellulose layers with colloidal silica particles was investigated by direct force measurements with the atomic force microscope (AFM). Upon approach, repulsive forces were found between the negatively charged silica particles and the cellulose surface. The forces were interpreted quantitatively in terms of electrostatic interactions due to overlap of diffuse layers originating from negatively charged carboxylic groups on the cellulose surface. The diffuse layer charge density of cellulose was estimated to be 0.80 mC/m2 at pH 9.5 and 0.21 mC/m2 at pH 4. The forces upon retraction are characterized by molecular adhesion events, whereby individual cellulose chains desorb from the probe surface. The retraction profiles are dominated by well-defined force plateaus, which correspond to single-chain desorption forces of 35-42 pN. We surmise that adsorption of cellulose to probe surfaces is dominated by nonelectrostatic forces, probably originating from hydrogen bonding. Electrostatic contributions to desorption force could be detected only at high pH, where the silica surface is highly charged.     

Probing Interactions within the synaptic DNA-SfiI complex by AFM force spectroscopy

SfiI belongs to a family of restriction enzymes that function as tetramers, binding two recognition regions for the DNA cleavage reaction. The SfiI protein is an attractive and convenient model for studying synaptic complexes between DNA and proteins capable of site-specific binding. The enzymatic action of SfiI has been very well characterized. However, the properties of the complex before the cleavage reaction are not clear. We used single-molecule force spectroscopy to analyze the strength of interactions within the SfiI-DNA complex. In these experiments, the stability of the synaptic complex formed by the enzyme and two DNA duplexes was probed in a series of approach-retraction cycles. In order to do this, one duplex was tethered to the surface and the other was tethered to the probe. The complex was formed by the protein present in the solution. An alternative setup, in which the protein was anchored to the surface, allowed us to probe the stability of the complex formed with only one duplex in the approach-retraction experiments, with the duplex immobilized at the probe tip. Both types of complexes are characterized by similar rupture forces. The stability of the complex was determined by measuring the dependence of rupture forces on force loading rates (dynamic force spectroscopy) and the results suggest that the dissociation reaction of the SfiI-DNA complex has a single energy barrier along the dissociation path. Dynamic force spectroscopy was instrumental in revealing the role of the 5 bp spacer region within the palindromic recognition site on DNA-SfiI in the stability of the complex. The data show that, although the change of non-specific sequence does not alter the position of the activation barrier, it changes values of the off rates significantly.     

Proteoglycan mechanics studied by single-molecule force spectroscopy of allotypic cell adhesion glycans

Early Metazoans had to evolve the first cell adhesion system addressed to maintaining stable interactions between cells constituting different individuals. As the oldest extant multicellular animals, sponges are good candidates to have remnants of the molecules responsible for that crucial innovation. Sponge cells associate in a species-specific process through multivalent calcium-dependent interactions of carbohydrate structures on an extracellular membrane-bound proteoglycan termed aggregation factor. Single-molecule force spectroscopy studies of the mechanics of aggregation factor self-binding indicate the existence of intermolecular carbohydrate adhesion domains. A 200-kDa aggregation factor glycan (g200) involved in cell adhesion exhibits interindividual differences in size and epitope content which suggest the existence of allelic variants. We have purified two of these g200 distinct forms from two individuals of the same sponge species. Comparison of allotypic versus isotypic g200 binding forces reveals significant differences. Surface plasmon resonance measurements show that g200 self-adhesion is much stronger than its binding to other unrelated glycans such as chondroitin sulfate. This adhesive specificity through multiple carbohydrate binding domains is a type of cooperative interaction that can contribute to explain some functions of modular proteoglycans in general. From our results it can be deduced that the binding strength/surface area between two aggregation factor molecules is comparable with that of focal contacts in vertebrate cells, indicating that strong carbohydrate-based cell adhesions evolved at the very start of Metazoan history.     

Atomic force spectroscopy-based study of antibody pesticide interactions for characterization of immunosensor surface

Development of immunobiosensor detector surfaces involves the immobilization of active antibodies on the capture surface without any significant loss of antigen binding activity. An atomic force microscope (AFM) was used to directly evaluate specific interactions between pesticides and antibodies on a biosensor surface. Oriented immobilization of antibodies against two herbicide molecules 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, on gold, was carried out to create the active immunobiosensor surfaces. The adhesive forces between immobilized antibodies and their respective antigens were measured by force spectroscopy using hapten-carrier protein functionalized AFM cantilevers. Relative functional affinity (avidity) measurements of the antibodies carried out prior to immobilization, well correlated with subsequent AFM force measurement observations. Analysis showed that immobilization had not compromised the reactivity of the surface immobilized antibody molecules for antigen nor was there any change in their relative quality with respect to each other. The utility of the immunoreactive surface was further confirmed using a Surface Plasmon Resonance (SPR) based detection system. Our study indicates that AFM can be utilized as a convenient immunobiosensing tool for confirming the presence and also assessing the strength of antibody-hapten interactions on biosensor surfaces under development.     

Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua

A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism.     

Probing effects of pH change on dynamic response of Claudin-2 mediated adhesion using single molecule force spectroscopy

Claudins belong to a large family of transmembrane proteins that localize at tight junctions (TJs) where they play a central role in regulating paracellular transport of solutes and nutrients across epithelial monolayers. Their ability to regulate the paracellular pathway is highly influenced by changes in extracellular pH. However, the effect of changes in pH on the strength and kinetics of claudin mediated adhesion is poorly understood. Using atomic force microscopy, we characterized the kinetic properties of homophilic trans-interactions between full length recombinant GST tagged Claudin-2 (Cldn2) under different pH conditions. In measurements covering three orders of magnitude change in force loading rate of 10²–10⁴ pN/s, the Cldn2/Cldn2 force spectrum (i.e., unbinding force versus loading rate) revealed a fast and a slow loading regime that characterized a steep inner activation barrier and a wide outer activation barrier throughout pH range of 4.5–8. Comparing to the neutral condition (pH 6.9), differences in the inner energy barriers for the dissociation of Cldn2/Cldn2 mediated interactions at acidic and alkaline environments were found to be < 0.65 k_BT, which is much lower than the outer dissociation energy barrier (> 1.37 k_BT). The relatively stable interaction of Cldn2/Cldn2 in neutral environment suggests that electrostatic interactions may contribute to the overall adhesion strength of Cldn2 interactions. Our results provide an insight into the changes in the inter-molecular forces and adhesion kinetics of Cldn2 mediated interactions in acidic, neutral and alkaline environments.     

Detecting Solvent-Driven Transitions of poly(A) to Double-Stranded Conformations by Atomic Force Microscopy

We report the results of direct measurements by atomic force microscopy of solvent-driven structural transitions within polyadenylic acid (poly(A)). Both atomic force microscopy imaging and pulling measurements reveal complex strand arrangements within poly(A) induced by acidic pH conditions, with a clear fraction of double-stranded molecules that increases as pH decreases. Among these complex structures, force spectroscopy identified molecules that, upon stretching, displayed two distinct plateau features in the force-extension curves. These plateaus exhibit transition forces similar to those previously observed in native double-stranded DNA (dsDNA). However, the width of the first plateau in the force-extension curves of poly(A) varies significantly, and on average is shorter than the canonical 70% of initial length corresponding to the B-S transition of dsDNA. Also, similar to findings in dsDNA, stretching and relaxing elasticity profiles of dspoly(A) at forces below the mechanical melting transition overlap but reveal hysteresis when the molecules are stretched above the mechanical melting transition. These results strongly suggest that under acidic pH conditions, poly(A) can form duplexes that are mechanically stable. We hypothesize that under acidic conditions, similar structures may be formed by the cellular poly(A) tails on mRNA.     

Measuring the interaction forces between protein inclusion bodies and an air bubble using an atomic force microscope

Interaction forces between protein inclusion bodies and an air bubble have been quantified using an atomic force microscope (AFM). The inclusion bodies were attached to the AFM tip by covalent bonds. Interaction forces measured in various buffer concentrations varied from 9.7 nN to 25.3 nN (+/- 4-11%) depending on pH. Hydrophobic forces provide a stronger contribution to overall interaction force than electrostatic double layer forces. It also appears that the ionic strength affects the interaction force in a complex way that cannot be directly predicted by DLVO theory. The effects of pH are significantly stronger for the inclusion body compared to the air bubble. This study provides fundamental information that will subsequently facilitate the rational design of flotation recovery system for inclusion bodies. It has also demonstrated the potential of AFM to facilitate the design of such processes from a practical viewpoint.     

The role of flexible tethers in multiple ligand-receptor bond formation between curved surfaces

Ligands mounted to surfaces via extensible tethers are present in nature and represent a growing class of molecules used to engineer adhesion in drug targeting, biosensing, self-assembling nanostructures, and in other biophysical research. Using a continuum approach with geometric and thermodynamic arguments, we derive a number of analytical expressions that relate key properties of single-tethered ligand-receptor interactions to multiple bond formation between curved surfaces. The theoretical predictions are in good agreement with measurements made with the surface forces apparatus. We establish that, when ligated, many tethers commonly used in biophysical research exhibit a discrete binding range that can be accurately measured with force spectroscopy. The distribution of bound ligated tethers is independent of the surfaces' interaction radius, R. The bridging force scales linearly with R, the tether's effective spring constant and grafting density, and with the ligand-receptor bond energy when the surfaces are in direct contact. These results are contrasted to bridging forces that evolve between plane-parallel geometries. Last, we show how our simple analytical reductions can be used to predict adhesive forces for STEALTH liposomes and other targeted and self-assembled nanoparticles.     

Interaction of bacterial endotoxins with chitosan. Effect of endotoxin structure, chitosan molecular mass, and ionic strength of the solution on the formation of the complex

The interaction of endotoxins of different structure (lipopolysaccharides (LPS) and lipopolysaccharide-protein complexes (LPPC)) with chitosan has been studied. It was shown that the mechanism of interaction is rather complicated and depends on the macromolecular organization of endotoxin as well as on the degree of polymerization of the chitosan. Chitosan with molecular mass of 20 kD reveals higher affinity to LPS than chitosan with molecular mass of 140 kD. Endotoxins with long O-specific chains can bind completely with chitosan with the formation of LPS-chitosan and LPPC-chitosan complexes with weight ratios between the original components of 1:1 and 1:5. When endotoxins with higher degree of hydrophobicity and short O-specific chains were mixed with chitosan, a part of the LPS remained unbound. The stability of the complexes formed depends on ionic strength. It was shown that, in addition to electrostatic forces, other types of forces take part in the formation of the complexes. A decrease in acute toxicity of various LPSs is observed on their binding with chitosans.     

Chitosan–bovine serum albumin complex formation: A model to design an enzyme isolation method by polyelectrolyte precipitation

Interactions between a model protein (bovine serum albumin—BSA) and the cationic polyelectrolyte, chitosan (Chi), have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It has been found that the conformation of the BSA does not change significantly during the chain interaction between BSA and chitosan forming the non-covalently linked complex. The effects of pH, ionic strength and anions which modify the water structure around BSA were evaluated in the chitosan–BSA complex formation. A net coulombic interaction force between BSA and Chi was found as the insoluble complex formation decreased after the addition of NaCl. Around 80% of the BSA in solution precipitates with the Chi addition. A concentration of 0.05% (w/v) Chi was necessary to precipitate the protein, with a stoichiometry of 6.9 g BSA/g Chi. No modification of the tertiary and secondary structure of BSA was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan proved to be a useful framework to isolate proteins with a slightly acid isoelectrical pH by means of precipitation.     

Physical gelation of chitosan in the presence of beta-glycerophosphate: the effect of temperature

When adding beta-glycerophosphate (beta-GP), a weak base, to chitosan aqueous solutions, the polymer remains in solution at neutral pH and room temperature, while homogeneous gelation of this system can be triggered upon heating. It is therefore one of the rare true physical chitosan hydrogels. In this study, physicochemical and rheological properties of chitosan solutions in the presence of acetic acid and beta-GP were investigated as a function of temperature in order to gain a better understanding of the gelation mechanisms. The gel structure formed at high temperature was only partially thermoreversible upon cooling to 5 degrees C because of the existence of remaining associations, confirmed by the spontaneous recovery of the gel after breakup at low temperature. Increasing temperature had no effect on the pH values of this system, while conductivity (and calculated ionic strength) increased. Values from the pH measurements were used to estimate the degree of protonation of each species as a function of temperature. The decreasing ratio of -NH3+ in chitosan and -OPO(O-)2 in beta-GP suggested reduced chitosan solubility along with a diminution of ionic interactions such as ionic bridging with increasing temperature. On the other hand, the increased ionic strength as a function of temperature, in the presence of beta-GP, enhanced screening of electrostatic repulsion and increased hydrophobic effect, resulting in favorable conditions for gel formation. Therefore, our study suggests that hydrophobic interactions and reduced solubility are the main driving force for chitosan gelation at high temperature in the presence of beta-GP.     

Dynamic response of glucagon/anti-glucagon pairs to pulling velocity and pH studied by atomic force microscopy

We used atomic force microscopy (AFM) to measure the unbinding force between antigen coupled to an AFM tip and antibody coated on the substrate surface. Dynamic responses of glucagon/anti-glucagon pairs with multiple pull-off steps to pH and pulling velocity were studied by AFM. Force-distance curves of a specific glucagon-anti-glucagon interaction system with mono-, di-, and multi-unbinding events were recorded, which may be attributed to a single, sequential or multiple breaking of interacting bond(s) between glucagon and anti-glucagon. We studied the dynamic response of glucagon-anti-glucagon pairs to various pulling velocities (16.7-166.7 nm/s). It was found that the mean value of the unbinding force was shifted toward higher values with increasing pulling velocity at each pH. This indicates that the friction force between glucagon and anti-glucagon may contribute to the unbinding force. Moreover, the dynamic response of glucagon-anti-glucagon pairs to pH (4-10) with different pulling velocities was studied. Within the acid range, the bond strength between the glucagon/anti-glucagon complex showed a rapid increase from pH 4 to 7 and reached a maximum (256.4+/-48.9 pN at 166.7 nm/s) at neutrality, followed by a sharp decrease with increasing pH (pH 7-10). This could be attributed to the conformational change that occurred in glucagon when the pH value in solution was varied from the reference level at neutrality. This study demonstrated that the pH dependence of multiple antigen-antibody bond-rupture forces could be measured by a force-based AFM biosensor. Unraveling the relationship between inter-molecular force and intra-molecular conformational change in acid, neutral, and alkaline environments may provide new directions for future application of force measurements by AFM in proteomics or in the development of a clinical cantilever-based mechanical biosensor.     

Atomic force microscopy study of the specific adhesion between a colloid particle and a living melanoma cell: Effect of the charge and the hydrophobicity of the particle surface

We investigated the effect of the charge and the hydrophobicity of drug delivery system (DDS) carriers on their specificity to living malignant melanoma B16F10 cells with the atomic force microscope. To model various nanoparticle DDS carriers, we used silica particles that were modified with silane coupling agents. We then measured the compression and decompression forces between the modified colloid probes and the living B16F10 cell in a physiological buffer as a function of their separation distances. The maximum adhesive force on decompression was related to the strength of the specificity of the DDS to the malignant cell. A comparison of the average maximum adhesive force of each functionality group surprisingly showed that negatively charged surfaces and hydrophobic modified surfaces all had similar low values. Additionally, we saw the unexpected result that there was no observable dependence on the degree of hydrophobicity of the probe surface to a B16F10 cell. Only the positively charged particle gave a strong adhesive force with the B16F10 cell. This indicated that DDS carriers with positive charges appeared to have the highest affinity for malignant melanoma cells and that the use of hydrophobic materials unexpectedly did not improve their affinity.     

Methods for reducing nonspecific interaction in antibody-antigen assay via atomic force microscopy

We developed a method to measure the rupture forces between antibody and antigen by atomic force microscopy (AFM). Previous studies have reported that in the measurement of antibody–antigen interaction using AFM, the specific intermolecular forces are often obscured by nonspecific adhesive binding forces between antibody immobilized cantilever and substrate surfaces on which antigen or nonantigen are fixed. Here, we examined whether detergent and nonreactive protein, which have been widely used to reduce nonspecific background signals in ordinary immunoassay and immunoblotting, could reduce the nonspecific forces in the AFM measurement. The results showed that, in the presence of both nonreactive protein and detergent, the rupture forces between anti-ferritin antibodies immobilized on a tip of cantilever and ferritin (antigen) on the substrate could be successfully measured, distinguishing from nonspecific adhesive forces. In addition, we found that approach/retraction velocity of the AFM cantilever was also important in the reduction of nonspecific adhesion. These insights will contribute to the detection of specific molecules at nanometer scale region and the investigation of intermolecular interaction by the use of AFM.     

Carbohydrate-carbohydrate interaction provides adhesion force and specificity for cellular recognition

The adhesion force and specificity in the first experimental evidence for cell-cell recognition in the animal kingdom were assigned to marine sponge cell surface proteoglycans. However, the question whether the specificity resided in a protein or carbohydrate moiety could not yet be resolved. Here, the strength and species specificity of cell-cell recognition could be assigned to a direct carbohydrate-carbohydrate interaction. Atomic force microscopy measurements revealed equally strong adhesion forces between glycan molecules (190-310 piconewtons) as between proteins in antibody-antigen interactions (244 piconewtons). Quantitative measurements of adhesion forces between glycans from identical species versus glycans from different species confirmed the species specificity of the interaction. Glycan-coated beads aggregated according to their species of origin, i.e., the same way as live sponge cells did. Live cells also demonstrated species selective binding to glycans coated on surfaces. These findings confirm for the first time the existence of relatively strong and species-specific recognition between surface glycans, a process that may have significant implications in cellular recognition.