Recent advances in sensing the inter-biomolecular interactions at the nanoscale - A comprehensive review of AFM-based force spectroscopy.

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.

Single-molecule force spectroscopy reveals structural differences of heparan sulfate chains during binding to vitronectin.

The syndecans represent an ongoing research field focused on their regulatory roles in normal and pathological conditions. The role of syndecans in cancer progression is well documented, implicating their importance in diagnosis and even proposing various potential cancer treatments. Thus, the characterization of the unbinding properties at the single-molecule level will appeal to their use as targets for therapeutics. In our study, syndecan-1 and syndecan-4 were measured during the interaction with the vitronectin HEP II binding site. Our findings show that syndecans are calcium ion dependent molecules that reveal distinct, unbinding properties indicating the alterations in the structure of heparan sulfate (HS) chains, possibly in the chain sequence or sulfation pattern. In this way, we suppose that HS chain affinity to extracellular matrix proteins may govern cancer invasion by altering the syndecans' ability to interact with cancer-related receptors present in the tumor microenvironment, thereby promoting the activation of various signaling cascades regulating tumor cell behavior.

Probing the recognition specificity of αVß1 integrin and syndecan-4 using force spectroscopy.

The knowledge on how cells interact with microenvironment is particularly important in understanding the interaction of cancer cells with surrounding stroma, which affects cell migration, adhesion, and metastasis. The main cell surface receptors responsible for the interaction with extracellular matrix (ECM) are integrins, however, they are not the only ones. Integrins are accompanied to other molecules such as syndecans. The role of the latter has not yet been fully established. In our study, we would like to answer the question of whether integrins and syndecans, possessing similar functions, share also similar unbinding properties. By using single molecule force spectroscopy (SMFS), we conducted measurements of the unbinding properties of αVß1 and syndecan-4 in the interaction with vitronectin (VN), which, as each ECM protein, possesses two binding sites specific to integrins and syndecans. The unbinding force and the kinetic off rate constant derived from SMFS describe the stability of single molecular complex. Obtained data show one barrier transition for each complex. The proposed model shows that the unbinding of αVß1 from VN proceeds before the unbinding of SDC-4. However, despite different unbinding kinetics, the access to both receptors is needed for cell growth and proliferation.

How strong are hydrogen bonds in the peptide model?

Detailed knowledge of intramolecular hydrogen bonds, including their nanomechanics, in a peptide secondary structure is crucial for understanding mechanisms of numerous biochemical processes. Single-molecule force spectroscopy has become a powerful tool to study directly the mechanical properties of single biopolymers and monitoring the hydrogen bonds. However, the interpretation of such experiments, due to their poor temporal resolution relative to the rate of intramolecular dynamics, requires the support of molecular simulations. In this work, we provide a methodology for determining the kinetic and energetic characteristics of hydrogen bonds in a template model of the protein secondary structure. Our approach, based on the steered molecular dynamics method, employs dynamic force spectroscopy calculations and uses two advanced theoretical models of force-induced unbinding. A systematic analysis of the simulated data with these models allowed for quantitative characterization of a single hydrogen bond in the α-helix of the AAKA(AEAAKA)5AC peptide model and detailed explanation of the mechanism of the α-helix unfolding. The methodology proposed here may be extended to other molecular structures stabilized by internal hydrogen bonds.

How Complex Is the Concanavalin A-Carboxypeptidase Y Interaction?

Lectin-carbohydrate interactions can be exploited in ultrasensitive biochemical recognition or medical diagnosis. For this purpose, besides the high specificity of the interactions, an appropriate methodology for their quantitative and detailed characterization is demanded. In this work, we determine the unbinding properties of the concanavalin A-carboxypeptidase Y complex, which is important for characterization of glycoproteins on the surface of biological cells. To achieve the goal, we have developed a methodology based on dynamic force spectroscopy measurements and two advanced theoretical models of force-induced unbinding. Our final results allowed excluding both, rebinding processes and the multibarrier character of the interaction potential, as possible explanations of the concanavalin A-carboxypeptidase Y unbinding mechanisms. Such characteristics as the position and height of the activation barrier and the force-free dissociation rate were determined. We hope our paper contributes to a better understanding of the unbinding processes in receptor-ligand complexes.

Bioengineering the spider silk sequence to modify its affinity for drugs.

BACKGROUND: Silk is a biocompatible and biodegradable material, able to self-assemble into different morphological structures. Silk structures may be used for many biomedical applications, including carriers for drug delivery. The authors designed a new bioengineered spider silk protein, EMS2, and examined its property as a carrier of chemotherapeutics. MATERIALS AND METHODS: To obtain EMS protein, the MS2 silk monomer (that was based on the MaSp2 spidroin of Nephila clavipes) was modified by the addition of a glutamic acid residue. Both bioengineered silks were produced in an Escherichia coli expression system and purified by thermal method. The silk spheres were produced by mixing with potassium phosphate buffer. The physical properties of the particles were characterized using scanning electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, and zeta potential measurements. The MTT assay was used to examine the cytotoxicity of spheres. The loading and release profiles of drugs were studied spectrophotometrically. RESULTS: The bioengineered silk variant, EMS2, was constructed, produced, and purified. The EMS2 silk retained the self-assembly property and formed spheres. The spheres made of EMS2 and MS2 silks were not cytotoxic and had a similar secondary structure content but differed in morphology and zeta potential values; EMS2 particles were more negatively charged than MS2 particles. Independently of the loading method (pre- or post-loading), the loading of drugs into EMS2 spheres was more efficient than the loading into MS2 spheres. The advantageous loading efficiency and release rate made EMS2 spheres a good choice to deliver neutral etoposide (ETP). Despite the high loading efficiency of positively charged mitoxantrone (MTX) into EMS2 particles, the fast release rate made EMS2 unsuitable for the delivery of this drug. A faster release rate from EMS2 particles compared to MS2 particles was observed for positively charged doxorubicin (DOX). CONCLUSION: By modifying its sequence, silk affinity for drugs can be controlled.

Unbinding Kinetics of Syndecans by Single-Molecule Force Spectroscopy.

Syndecans are transmembrane proteoglycans that, together with integrins, control cell interactions with extracellular matrix components. Despite structural similarities between all members of the syndecan family, their specific attachment to extracellular matrix proteins is defined by heparan and chondroitin chains. We postulate various unbinding kinetics for each type of single syndecan complex. Force spectroscopy data, recorded by atomic force microscope, were analyzed using two theoretical approaches describing force-induced unbinding, authored by Bell-Evans and Dudko-Hummer-Szabo. Our results reveal distinct unbinding pathways dependent on the syndecan family member. Syndecan-1 unbinds by passing over two energy barriers, inner and outer. Syndecan-4 unbinds by crossing over only one energy barrier. It has already been reported that both syndecans bear heparan chains that are structurally indistinguishable. Our finding reveals that unbinding of single syndecan complexes is family-member-dependent. Distinct unbinding pathways can be attributed to structural differences of heparan and chondroitin chains.

Effect of the Chain Length and Temperature on the Adhesive Properties of Alkanethiol Self-Assembled Monolayers.

Stable and hydrophobic self-assembled monolayers of alkanethiols are promising materials for use as lubricants in microdevices and nanodevices. We applied high-rate dynamic force spectroscopy measurements to study in detail the influence of the chain length and temperature on the adhesion between methyl-terminated thiol monolayers and a silicon nitride tip. We used the Johnson-Kendall-Roberts model to calculate the number of molecules in adhesive contact and then the Dudko-Hummer-Szabo model to extract the information about the position and the height of the activation barrier per single molecule. Both parameters were determined and analyzed in the temperature range from 25 to 65 °C for three thiols: 1-decanethiol (measured previously), 1-tetradecanethiol, and 1-hexadecanethiol. We associate the increase of the activation barrier parameters versus the chain length with lower stiffness of longer molecules and higher effectiveness of adhesive bond formation. However, we relate the thermal changes of the parameters rather to rearrangements of molecules than to the direct influence of temperature on the adhesive bonds.

Influence of Temperature on the Nanoadhesion of a Methyl-Terminated Thiol Monolayer: A New Insight with High-Rate Dynamic Force Spectroscopy.

Alkanethiols form stable, homogeneous, and well-organized self-assembled monolayers and can be used as lubricants in micro- and nanodevices when sufficiently hydrophobic and resistant to sudden temperature changes. In this paper, we demonstrate a new analysis method which provides a deep physical insight into adhesive interactions and their temperature dependencies at the single molecule level. We have focused on the adhesion between a silicon nitride tip and a 1-decanethiol self-assembled monolayer in the temperature range from 25 to 85 °C. We performed dynamic force spectroscopy measurements and applied theoretical models of adhesive-mechanical interactions and thermally activated unbinding to obtain detailed information on the adhesive interactions. The parameters of the interaction potential describing a single adhesive bond were calculated, and their temperature dependence was discussed. Although the changes of the adhesion force versus temperature are significant and nonmonotonic, the energy of the activation barrier of a single adhesive bond appears temperature independent. We attribute observed changes in the position of the activation barrier to the interplay between the rupture and rebinding of adhesive bonds, as well as to thermal reorganization, in particular the change of the tilt angle of thiol molecules in the self-assembled monolayer.

Nanoadhesion on rigid methyl-terminated biphenyl thiol monolayers: a high-rate dynamic force spectroscopy study.

Nanoadhesion on a self-assembled monolayer of 4-methyl-4'-mercaptobiphenyl is measured using a modified atomic force microscope. The dependence of the adhesion force on the loading rate is analyzed with the Dudko-Hummer-Szabo model, and the kinetic and interaction potential parameters for a single terminal group are extracted. The energy and location of the activation barrier suggest that the adhesion is dominated by van der Waals dispersion forces. The humidity effect on the nanoadhesion is also studied. The results are compared with previously measured values for methyl-terminated alkane thiols and the influence of the thiol rigidity on the adhesion force is discussed.

Effect of humidity on nanoscale adhesion on self-assembled thiol monolayers studied by dynamic force spectroscopy.

The adhesion force between silicon nitride tips of an atomic force microscope and different self-assembled thiol monolayers (SAMs) was measured at different loading rates and humidity. SAMs were formed from HS(CH(2))(n)CH(3) with n = 6, 8, 9, 10, 15 and HS(CH(2))(n)OH with n = 6, 9, 11, 16. With a special setup, the loading rate could be increased to 10(7) nN s(-1). For the interaction with two-dimensional crystalline CH(3)-terminated SAMs (n > or = 8), two regimes can be distinguished. At loading rates below 10(4)-10(5) nN s(-1), the adhesion force increased proportional to the logarithm of the loading rate. Adhesion is most likely dominated by van der Waals attraction. At higher loading rates, the adhesion forces increased steeper with the logarithm of the loading rate. The specific process limiting separation is not yet identified. On OH-terminated SAMs, the adhesion force was approximately 6 times higher than on the CH(3)-terminated SAMs, even at low humidity. This can partially, but not fully, be explained by hydrogen bridges forming between the hydroxyl groups of the monolayer and silanol groups of the tip. For relative humidity above 10%, the capillary force further increased the adhesion force, which reached a maximum at values of relative humidity between 40% and 80%. Adhesion force versus loading rate (F(ad) versus r(F)) curves increased roughly linearly over the whole range of loading rates. The slope depended on the humidity, and it is correlated with the absolute strength of the capillary force.

Quantitative characterization of nanoadhesion by dynamic force spectroscopy.

We present a method for the characterization of adhesive bonds formed in nanocontacts. Using a modified atomic force microscope, the nanoadhesion between a silicon nitride tip and a self-assembled monolayer of 1-nonanethiol on gold(111) was measured at different loading rates. Adhesion force-versus-loading rate curves could be fitted with two logarithmic terms, indicating a two step (two energy barrier) process. The application of the Bell-Evans model and classical contact mechanics allows the extraction of quantitative information about the effective adhesion potential and characterization of the different components contributing to nanoadhesion.

Fluorescence polarization studies of B-phycoerythrin oriented in polymer film.

Polarized steady-state fluorescence and fluorescence excitation spectra as well as time-resolved fluorescence for B-phycoerythrin (B-PE) from red algae, Porphyridium cruentum, embedded in polyvinyl stretched films were measured. The lifetimes of polarized fluorescence were analyzed using exponential components and fractal models. The interactions between various chromophores of the pigment-protein complexes investigated were discussed. The anisotropy of fluorescence excitation spectra differs from the anisotropy of absorption spectra and depends on the wavelength of observation. This shows that differently oriented chromophores take part in various paths of excitation energy transfer (ET) or change their excitation into heat with various efficiencies (or both). Also, analysis of time-resolved fluorescence measured in various spectral regions gives different polarized components of emission. Fractal analysis of lifetimes, done under supposition of the Foerster resonance ET mechanism, suggests different arrangements of energy donors and acceptors for molecules absorbing in different spectral regions. It shows that several fractions of differently oriented "forms" of chromophores exhibiting different spectral properties occur in B-PE complexes. Small changes in the orientation of the chromophores can be followed by modification of the path of excitation energy migration. Based on the results obtained a new reorientational mechanism of the State 1 --> State 2 transition was proposed: Even small conformational modifications of biliproteins, which could be caused in vivo by the change in the conditions of preillumination of bacteria, are able to modify the path of excitation ET. Such a reorientation may be responsible for the change in the partition of biliprotein excitation energy between photosystem II (PSII) and PSI (State 1 --> State 2 transition). The proposed mechanism needs further verification by the investigation of whole bacteria cells.