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.     

Direct observation of active protein folding using lock-in force spectroscopy

Direct observation of the folding of a single polypeptide chain can provide important information about the thermodynamic states populated along its folding pathway. In this study, we present a lock-in force-spectroscopy technique that improves resolution of atomic-force microscopy force spectroscopy to 400 fN. Using this technique we show that immunoglobulin domain 4 from Dictyostelium discoideum filamin (ddFLN4) refolds against forces of ∼4 pN. Our data show folding of this domain proceeds directly from an extended state and no thermodynamically distinct collapsed state of the polypeptide before folding is populated. Folding of ddFLN4 under load proceeds via an intermediate state. Three-state folding allows ddFLN4 to fold against significantly larger forces than would be possible for a mere two-state folder. We present a general model for protein folding kinetics under load that can predict refolding forces based on chain-length and zero force refolding rate.     

Direct discrimination between models of protein activation by single-molecule force measurements

The limitations imposed on the analyses of complex chemical and biological systems by ensemble averaging can be overcome by single-molecule experiments. Here, we used a single-molecule technique to discriminate between two generally accepted mechanisms of a key biological process--the activation of proteins by molecular effectors. The two mechanisms, namely induced-fit and population-shift, are normally difficult to discriminate by ensemble approaches. As a model, we focused on the interaction between the nuclear transport effector, RanBP1, and two related complexes consisting of the nuclear import receptor, importin beta, and the GDP- or GppNHp-bound forms of the small GTPase, Ran. We found that recognition by the effector proceeds through either an induced-fit or a population-shift mechanism, depending on the substrate, and that the two mechanisms can be differentiated by the data.     

Direct measurement of local chromatin fluidity using optical trap modulation force spectroscopy

Chromatin assembly is condensed by histone tail-tail interactions and other nuclear proteins into a highly compact structure. Using an optical trap modulation force spectroscopy, we probe the effect of tail interactions on local chromatin fluidity. Chromatin fibers, purified from mammalian cells, are tethered between a microscope coverslip and a glass micropipette. Mechanical unzipping of tail interactions, using the micropipette, lead to the enhancement of local fluidity. This is measured using an intensity-modulated optically trapped bead positioned as a force sensor on the chromatin fiber. Enzymatic digestion of the histone tail interactions of tethered chromatin fiber also leads to a similar increase in fluidity. Our experiments show that an initial increase in the local fluidity precedes chromatin decompaction, suggesting possible mechanisms by which chromatin-remodeling machines access regulatory sites.     

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.     

Nonspecific interactions in AFM force spectroscopy measurements

Sample-probe contact duration (dwell time) and loading force are two important parameters for the atomic force microscopy (AFM) force spectroscopy measurements of ligand-receptor interaction. A prolonged contact time may be required to initiate ligand-receptor binding as a result of slow on-rate kinetics or low reactant density. In general, increasing contact duration promotes nonspecific interactions between the substrate and the functionalized cantilever and, thus, masking the detection of the specific interactions. To reduce the nonspecific interactions in AFM force measurements requiring extended substrate-probe contact, we investigated the interaction of bovine serum albumin (BSA)-functionalized cantilever with BSA-coated glass, polyethylene glycol (PEG)-functionalized glass, Pluronic-treated Petri dishes and agarose beads. The frequency of nonspecific interaction between the BSA-functionalized cantilever and the different samples increased with loading force and dwell time. This increase in nonspecific adhesion can be attributed to the interaction mediated by forced unfolding of BSA. By reducing the loading force, the contact duration of the AFM probe with an agarose bead can be extended to a few minutes without nonspecific adhesion.     

Direct mechanical force measurements during the migration of Dictyostelium slugs using flexible substrata

We use the flexible substrate method to study how and where mechanical forces are exerted during the migration of Dictyostelium slugs. This old and contentious issue has been left poorly understood so far. We are able to identify clearly separate friction forces in the tip and in the tail of the slug, traction forces mostly localized in the inner slug/surface contact area in the prespore region and large perpendicular forces directed in the outward direction at the outline of contact area. Surprisingly, the magnitude of friction and traction forces is decreasing with slug velocity indicating that these quantities are probably related to the dynamics of cell/substrate adhesion complexes. Contrary to what is always assumed in models and simulations, friction is not of fluid type (viscous drag) but rather close to solid friction. We suggest that the slime sheath confining laterally the cell mass of the slug experiences a tension that in turn is pulling out the elastic substrate in the direction tangential to the slug profile where sheath is anchored. In addition, we show in the appendix that the iterative method we developed is well adapted to study forces over large and continuous fields when the experimental error is sufficiently low and when the plane of recorded bead deformations is close enough to the elastomer surface, requirements fulfilled in this experimental study of Dictyostelium slugs.     

Pulling geometry-induced errors in single molecule force spectroscopy measurements

In AFM-based single molecule force spectroscopy, it is tacitly assumed that the pulling direction coincides with the end-to-end vector of the molecule fragment being stretched. By systematically varying the position of the attachment point on the substrate relative to the AFM tip, we investigate empirically and theoretically the effect of the pulling geometry on force-extension characteristics of double-stranded DNA. We find that increasing the pulling angle can significantly lower the force of the characteristic overstretching transition and increase the width of the plateau feature beyond the canonical 70%. These effects, when neglected, can adversely affect the interpretation of measured force-extension relationships. We quantitatively evaluate force and extension errors originating from this "pulling angle effect" and stress the need to correct the pulling geometry when stretching rigid molecules with an AFM.     

Subpiconewton dynamic force spectroscopy using magnetic tweezers

We introduce a simple method for dynamic force spectroscopy with magnetic tweezers. This method allows application of subpiconewton force and twist control by calibration of the applied force from the height of the magnets. Initial dynamic force spectroscopy experiments on DNA molecules revealed a large hysteresis that is caused by viscous drag on the magnetic bead and will conceal weak interactions. When smaller beads are used, this hysteresis is sufficiently reduced to reveal intramolecular interactions at subpiconewton forces. Compared with typical quasistatic force spectroscopy, a significant reduction of measurement time is achieved, allowing the real-time study of transient structures and reaction intermediates. As a proof of principle, nucleosome-nucleosome interactions on a subsaturated chromatin fiber were analyzed.     

Single molecule force spectroscopy in biology using the atomic force microscope

The importance of forces in biology has been recognized for quite a while but only in the past decade have we acquired instrumentation and methodology to directly measure interactive forces at the level of single biological macromolecules and/or their complexes. This review focuses on force measurements performed with the atomic force microscope. A general introduction to the principle of action is followed by review of the types of interactions being studied, describing the main results and discussing the biological implications.     

Atomic force microscopy measurements. Measurements of binding strength between a single pair of molecules in physiological solutions

Atomic force microscopy (AFM) measurements of intermolecular binding strength between a single pair of complementary cell adhesion molecules in physiological solutions provided the first quantitative evidence for their cohesive function. This novel AFM based nanobiotechnology opens a molecular mechanic approach for studying structure to function related properties of any type of individual biological macromolecules. The presented example of Porifera cell adhesion glyconectin proteoglycans showed that homotypic carbohydrate to carbohydrate interactions between two primordial proteogylycans can hold the weight of 1600 cells. Thus, glyconectin type carbohydrates, as the most peripheral cell surface molecules of sponges (today’s simplest living Metazoa), are proposed to the primary cell adhesive molecules essential for the evolution of the multicellularity.     

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.     

Spatially resolved force spectroscopy of biological surfaces using the atomic force microscope

The spatial distribution of intermolecular forces governs macromolecular interactions. The atomic force microscope, a relatively new tool for investigating interaction forces between nanometer-scale objects, can be used to produce spatially resolved maps of the surface or material properties of a sample; these include charge density, adhesion and stiffness, as well as the force required to break specific ligand-receptor bonds. Maps such as these will provide fundamental insights into biological structure and will become an important tool for characterizing technologically important biological systems.     

Dynamic force measurements of avidin-biotin and streptavdin-biotin interactions using AFM

Using atomic force microscopy (AFM) we performed dynamic force measurements of the adhesive forces in two model systems: avidin-biotin and streptavidin-biotin. In our experiments we used glutaraldehyde for immobilization of (strept)avidin on the tip and biotin on the sample surface. Such interface layers are more rigid than those usually reported in the literature for AFM studies, when (strept)avidin is coupled with biotinylated bovine albumin and biotin with agarose polymers. We determined the dependence of the rupture forces of avidin-biotin and streptavidin-biotin bonds in the range 300-9600 pN/s. The slope of a semilogarithmic plot of this relation changes at about 1700 pN/s. The existence of two different regimes indicates the presence of two activation barriers of these complexes during the dissociation process. The dissociation rates and activation energy barriers, calculated from the Bell model, for the avidin-biotin and streptavidin-biotin interactions are similar to each other for loading rates > 1700 pN/s but they are different from each other for loading rates < 1700 pN/s. In the latter case, the dissociation rates show a higher stability of the avidin-biotin complex than the streptavidin-biotin complex due to a larger outer activation barrier of 0.8 k(B)T. The bond-rupture force is about 20 pN higher for the avidin-biotin pair than for the streptavidin-biotin pair for loading rates < 1700 pN/s. These two experimental observations are in agreement with the known structural differences between the biotin binding pocket of avidin and of streptavidin.     

Detection of phenolic compounds using impedance spectroscopy measurements

Langmuir-Blodgett (LB) and layer-by-layer films (LbL) of a PPV (p-phenylenevinylene) derivative, an azo compound and tetrasulfonated phthalocyanines were successfully employed as transducers in an “electronic tongue” system for detecting trace levels of phenolic compounds in water. The choice of the materials was based on their distinct electrical natures, which enabled the array to establish a fingerprint of very similar liquids. Impedance spectroscopy measurements were taken in the frequency range from 10 Hz to 1 MHz, with the data analysed with principal component analysis (PCA). The sensing units were obtained from five-layer LB films of (poly[(2-methoxy-5-n-hexyloxy)-p-phenylenevinylene]), OC₁OC₁₈-PPV (poly(2-methoxy,5-(n-octadecyl)-p-phenylenevinylene)), DR (HEMA-co-DR13MA (poly-(hydroxyethylmethacrylate-co-[4′-[[2-(methacryloyloxy)-ethyl]ethylamino]-2-chloro-4-nitroazobenzene]))) and five-bilayer LbL films of tetrasulfonated metallic phthalocyanines deposited onto gold interdigitated electrodes. The sensors were immersed into phenol, 2-chloro-4-methoxyphenol, 2-chlorophenol and 3-chlorophenol (isomers) solutions at 1 × 10⁻⁹ mol L⁻¹, with control experiments carried out in ultra pure water. Samples could be distinguished if the principal component analysis (PCA) plots were made with capacitance values taken at 10³ Hz, which is promising for detection of trace amounts of phenolic pollutants in natural water.     

Role of multiple bonds between the single cell adhesion molecules, nectin and cadherin, revealed by high sensitive force measurements

Nectins and cadherins, members of cell adhesion molecules (CAMs), are the primary mediators for various types of cell-cell junctions. Here, intermolecular force microscopy (IFM) with force sensitivity at sub-picoNewtons is used to characterize the extracellular trans-interactions between paired nectins and paired cadherins at the single molecule level. Three and four different bound states between paired nectins and paired cadherins are, respectively, identified and characterized based on bond strength distributions where each bound state has a unique lifetime and bond length. The results indicate that multiple domains of nectins act uncooperatively, as a zipper-like multiply bonded system whereas those of cadherins act cooperatively, as a parallel-like multiply bonded system, consistent with a "fork initiation and zipper" hypothesis for the formation of cell-cell adhesion. The observed dynamic properties among multiple bonds are expected to be advantageous such that nectins search adaptively in the cell-cell exploratory recognition process while cadherins slowly stabilize in the cell-cell zippering process.     

Direct comparison of muscle force predictions using linear and nonlinear programming

Estimating forces in muscles and joints during locomotion requires formulations consistent with available methods of solving the indeterminate problem. Direct comparisons of results between differing optimization methods proposed in the literature have been difficult owing to widely varying model formulations, algorithms, input data, and other factors. We present an application of a new optimization program which includes linear and nonlinear techniques allowing a variety of cost functions and greater flexibility in problem formulation. Unified solution methods such as the one demonstrated here, offer direct evaluations of such factors as optimization criteria and constraints. This unified method demonstrates that nonlinear formulations (of the sort reported) allow more synergistic activity and in contrast to linear formulations, allow antagonistic activity. Concurrence of EMG activity and predicted forces is better with nonlinear predictions than linear predictions. The prediction of synergistic and antagonistic activity expectedly leads to higher joint force predictions. Relaxation of the requirement that muscles resolve the entire intersegmental moment maintains muscle synergism in the nonlinear formulation while relieving muscle antagonism and reducing the predicted joint contact force. Such unified methods allow more possibilities for exploring new optimization formulations, and in comparing the solutions to previously reported formulations.     

Quantitative measurements of force and displacement using an optical trap.

We combined a single-beam gradient optical trap with a high-resolution photodiode position detector to show that an optical trap can be used to make quantitative measurements of nanometer displacements and piconewton forces with millisecond resolution. When an external force is applied to a micron-sized bead held by an optical trap, the bead is displaced from the center of the trap by an amount proportional to the applied force. When the applied force is changed rapidly, the rise time of the displacement is on the millisecond time scale, and thus a trapped bead can be used as a force transducer. The performance can be enhanced by a feedback circuit so that the position of the trap moves by means of acousto-optic modulators to exert a force equal and opposite to the external force applied to the bead. In this case the position of the trap can be used to measure the applied force. We consider parameters of the trapped bead such as stiffness and response time as a function of bead diameter and laser beam power and compare the results with recent ray-optic calculations.     

Analysis of DNA and Zinc finger interactions using mechanical force spectroscopy

The unbinding force of Zif268-DNA complex has been studied by atomic force microscopy (AFM). DNA and Zif268 were covalently immobilized on the surfaces of an AFM tip and glass substrate, respectively. Confocal microscopy was used to confirm the successful immobilization of DNA. Because of the complexity of the protein-DNA interaction, parallel experiments were designed to discriminate specific interactions. For such experiments, a typical unbinding force of a single Zif268-DNA complex (approx 550 pN at 40 nN/s force loading rate) was evaluated.