Unbinding Process Research Articles

Molecular Force Balance Measurements Reveal that Double-Stranded DNA Unbinds Under Force in Rate-Dependent Pathways

Strand separation of double-stranded DNA is a crucial step for essential cellular processes such as recombination and transcription. By means of a molecular force balance, we have analyzed the impact of different pulling directions and different force-loading rates on the unbinding process of short double-stranded DNA. At loading rates above 9×105pN/s, we found a marked difference in rupture probability for pulling the duplex in 3′-3′ direction compared to a 5′-5′ direction, indicating different unbinding pathways. We propose a mechanism by which unbinding at low loading rates is dominated by nondirectional thermal fluctuations, whereas mechanical properties of the DNA become more important at high loading rates and reveal the asymmetry of the phosphoribose backbone. Our model explains the difference of 3′-3′ and 5′-5′ unbinding as a kinetic process, where the loading rate exceeds the relaxation time of DNA melting bubbles.

The antigen–antibody unbinding process through steered molecular dynamics of a complexof an Fv fragment and lysozyme

We have investigated the antigen–antibody unbinding process using steered moleculardynamics (SMD) simulations. We focus on a complex system consisting of an Fv fragmentof an antibody molecule and a lysozyme as an antigen molecule. The Fv fragment consistsof a VL and VH chain. The results show that the VH chain is unbound earlier than theVL chain, which is confirmed by the ensemble average of the distance profileobtained from 40 unbinding trajectories. The use of lysozyme as an antigen moleculeinstead of a small hapten molecule reveals the fact that the induced fit, estimatedby the deformation accompanying the unbinding process, is more noticeable forthe antigen molecule than for the antibody molecule. The SMD also reveals thenon-Gaussian distribution of maximum force necessary for the unbinding process.

Unbinding of Nicotine from the Acetylcholine Binding Protein: Steered Molecular Dynamics Simulations

The ligand binding/unbinding process is critical to our understanding of the pharmacology of both the nicotinic acetylcholine receptor (nAChR) and the acetylcholine binding protein (AChBP). Steered molecular dynamics simulations were performed to learn about the unbinding process of the full agonist nicotine. Three different pulling models were designed to investigate the possible binding/unbinding pathways: radial and tangent models, and also a mixed model. Of the three, the tangent pulling model finally failed to dissociate nicotine from the ligand binding pocket. The efficiency of the pulling force profiles was superior, and the opening of the C-loop was smaller in the mixed pulling model than that in the radial model. The most favorable pathway for the cholinergic agonist nicotine to enter or leave the binding pocket is through the principal binding side, following a curvilinear track. Noticeably, it has been seen that the unbinding of the nicotine is concomitant with a global rotation of the protein-ligand complex which could be caused by the interactions of the ligand with protein at the tangent direction.

Study on the mechanism of the BtuF periplasmic-binding protein for vitamin B 12

BtuF is the periplasmic binding protein (PBP) that binds vitamin B 12 and delivers it to the periplasmic surface of the ABC transporter BtuCD. PBPs generally exhibit considerable conformational changes during ligand binding process, however, BtuF belongs to a subclass of PBPs that, doesn't show such behavior on the basis of the crystal structures. Employing steered molecular dynamics on the B 12-bound BtuF, we investigated the energetics and mechanism of BtuF. A potential of mean force along the postulated vitamin B 12 unbinding pathway was constructed through Jarzynski's equality. The large free energy differences of the postulated B 12 unbinding process suggests the B 12-bound structure is in a stable closed state and some conformation changes may be necessary to the B 12 unbinding. From the result of the principal component analysis, we found the BtuF-B 12 complex shows clear opening-closing and twisting motion tendencies which may facilitate the unbinding of B 12 from the binding pocket. The intrinsic flexibility of BtuF was also explored, and it's suggested the Trp44-Gln45 pair, which is situated at the mouth of the B 12 binding pocket, may act as a gate in the B 12 binding and unbinding process.

Mapping the Number of Molecules and Brightness in the Laser Scanning Microscope

We describe a technique based on moment-analysis for the measurement of the average number of molecules and brightness in each pixel in fluorescence microscopy images. The average brightness of the particle is obtained from the ratio of the variance to the average intensity at each pixel. To obtain the average number of fluctuating particles, we divide the average intensity at one pixel by the brightness. This analysis can be used in a wide range of concentrations. In cells, the intensity at any given pixel may be due to bright immobile structures, dim fast diffusing particles, and to autofluorescence or scattering. The total variance is given by the variance of each of the above components in addition to the variance due to detector noise. Assuming that all sources of variance are independent, the total variance is the sum of the variances of the individual components. The variance due to the particles fluctuating in the observation volume is proportional to the square of the particle brightness while the variance of the immobile fraction, the autofluorescence, scattering, and that of the detector is proportional to the intensity of these components. Only the fluctuations that depend on the square of the brightness (the mobile particles) will have a ratio of the variance to the intensity >1. Furthermore, changing the fluorescence intensity by increasing the illumination power, distinguishes between these possible contributions. We show maps of molecular brightness and number of cell migration proteins obtained using a two-photon scanning microscope operating with a photon-counting detector. These brightness maps reveal binding dynamics at the focal adhesions with pixel resolution and provide a picture of the binding and unbinding process in which dim molecules attach to the adhesions or large molecular aggregates dissociate from adhesion.

A rational method for probing macromolecules dissociation: the antibody-hapten system

The unbinding process of a protein-ligand complex of major biological interest was investigated by means of a computational approach at atomistic classical mechanical level. An energy minimisation-based technique was used to determine the dissociation paths of the system by probing only a relevant set of generalized coordinates. The complex problem was reduced to a low-dimensional scanning along a selected distance between the protein and the ligand. Orientational coordinates of the escaping fragment (the ligand) were also assessed in order to further characterise the unbinding. Solvent effects were accounted for by means of the Poisson–Boltzmann continuum model. The corresponding dissociation time was derived from the calculated barrier height, in compliance with the experimentally reported Arrhenius-like behaviour. The computed results are in good agreement with the available experimental data.

On molecular recognition of an uranyl chelate by monoclonal antibodies

The energy landscape of the uranyl (UO2) chelate dissociated from a monoclonal antibody U08S was investigated using dynamic force spectroscopy (DFS). The uranyl ion (UO2(2+)) is chelated with the ligand dicarboxy-phenanthroline (DCP). The monoclonal antibody U08S was raised against UO2-DCP and does not cross-react with DCP alone. The results of plotting the most probable force against the logarithm of the loading rate show two distinguished values of slopes of multiple fitting lines, as observed in our previous study on that system with monoclonal antibody U04S (Odorico et al., 2007a. Biophys. J. 93: 645-654.). It indicates an unbinding process undergoing at least two activation states. We have generated the histogram of unbinding events with respect to the composite stiffness of the complex between the protein and the uranyl compound. Combining the model of Bell and Evans with that of Williams, we have estimated the number of parallel bonds involved in the unbinding process and determined the value of stiffness for individual bonds. We propose that the uranyl compound binds to the two antibodies U04S and U0c at structurally equivalent locations and forms the interaction with similar coordination modes. In addition, the unbinding process goes through two steps; the first weakens the bonding of the central metal with AspL50 of the antibody and the second breaks other non-bonded interactions of the compound with the antibody.

Affinity-Matured Recombinant Antibody Fragments Analyzed by Single-Molecule Force Spectroscopy

For many applications, antibodies need to be engineered toward maximum affinity. Strategies are in demand to especially optimize this process toward slower dissociation rates, which correlate with the (un)binding forces. Using single-molecule force spectroscopy, we have characterized three variants of a recombinant antibody single-chain Fv fragment. These variants were taken from different steps of an affinity maturation process. Therefore, they are closely related and differ from each other by a few mutations only. The dissociation rates determined with the atomic force microscope differ by one order of magnitude and agree well with the values obtained from surface plasmon resonance measurements. However, the effective potential width of the binding complexes, which was derived from the dynamic force spectroscopy measurements, was found to be the same for the different mutants. The large potential width of 0.9 nm indicates that both the binding pocket and the peptide deform significantly during the unbinding process.

Dynamic Force Spectroscopy of the Specific Interaction between the PDZ Domain and Its Recognition Peptides

To characterize the molecular basis of specific interactions of PDZ proteins, dynamic force spectroscopy (DFS) for the PDZ protein Tax-interacting protein-1 (TIP-1) and its recognition peptide (PDZ-pep) derived from beta-catenin was performed using an atomic force microscope (AFM), together with measurement of thermodynamic and kinetic parameters using surface plasmon resonance (SPR). The unbinding force of this pair was measured under different conditions of AFM tip-retraction velocity. The relationship between the unbinding force and the logarithmic force-loading rate, that is, the dynamic force spectrum, exhibited two different rate regimes, for each of which the forces increased linearly with the force-loading rate. On the basis of the theoretical treatment of the Bell-Evans model, the positions of two different activation barriers in the reaction coordinate and dissociation rate constants in each barrier were evaluated from slopes and x-intercepts of the two linear regimes (first barrier: 0.04 nm and 1.10 x 10 s(-1); second barrier: 0.21 nm and 2.77 x 10(-2) s(-1), respectively). Although two-step unbinding kinetics between TIP-1 and PDZ-pep was suggested from the DFS analysis, SPR results showed single-step dissociation kinetics with a rate constant of 2.89 x 10(-1) s(-1). Different shapes of the free energy profile of the unbinding process were deduced from each result of DFS and SPR. The reason for such topographic differences in the energy landscape is discussed in relation to the differences in the pathways of forced unbinding and spontaneous dissociation.

Energy Landscape of Aptamer/Protein Complexes Studied by Single‐Molecule Force Spectroscopy

Aptamers are single-stranded nucleic acid molecules selected in vitro to bind to a variety of target molecules. Aptamers bound to proteins are emerging as a new class of molecules that rival commonly used antibodies in both therapeutic and diagnostic applications. With the increasing application of aptamers as molecular probes for protein recognition, it is important to understand the molecular mechanism of aptamer-protein interaction. Recently, we developed a method of using atomic force microscopy (AFM) to study the single-molecule rupture force of aptamer/protein complexes. In this work, we investigate further the unbinding dynamics of aptamer/protein complexes and their dissociation-energy landscape by AFM. The dependence of single-molecule force on the AFM loading rate was plotted for three aptamer/protein complexes and their dissociation rate constants, and other parameters characterizing their dissociation pathways were obtained. Furthermore, the single-molecule force spectra of three aptamer/protein complexes were compared to those of the corresponding antibody/protein complexes in the same loading-rate range. The results revealed two activation barriers and one intermediate state in the unbinding process of aptamer/protein complexes, which is different from the energy landscape of antibody/protein complexes. The results provide new information for the study of aptamer-protein interaction at the molecular level.

Simple Models for Extracting Mechanical Work from the ATP Hydrolysis Cycle

According to the binding-zipper model, the RecA class of ATPase motors converts chemical energy into mechanical force by the progressive annealing of hydrogen bonds between the nucleotide and the catalytic pocket. The role of hydrolysis is to weaken the binding of products, allowing them to be released so that the cycle can repeat. Molecular dynamics can be used to study the unbinding process, but the binding process is more complex, so that inferences about it are made indirectly from structural, mutation, and biochemical studies. Here we present a series of models of varying complexity that illustrate the basic processes involved in force production during ATP binding. These models reveal the role of solvent and geometry in determining the amount of mechanical work that can be extracted from the binding process.

Potentials of Mean Force for Acetylcholine Unbinding from the Alpha7 Nicotinic Acetylcholine Receptor Ligand-Binding Domain

The nicotinic acetylcholine receptor is a prototype ligand-gated ion channel that mediates signal transduction in the neuromuscular junctions and other cholinergic synapses. The molecular basis for the energetics of ligand binding and unbinding is critical to our understanding of the pharmacology of this class of receptors. Here, we used steered molecular dynamics to investigate the unbinding of acetylcholine from the ligand-binding domain of human alpha7 nicotinic acetylcholine receptor along four different predetermined pathways. Pulling forces were found to correlate well with interactions between acetylcholine and residues in the binding site during the unbinding process. From multiple trajectories along these unbinding pathways, we calculated the potentials of mean force for acetylcholine unbinding. Four available methods based on Jarzynski's equality were used and compared for their efficiencies. The most probable pathway was identified to be along a direction approximately parallel to the membrane. The derived binding energy for acetylcholine was in good agreement with that derived from the experimental binding constant for acetylcholine binding protein, but significantly higher than that for the complete human alpha7 nicotinic acetylcholine receptor. In addition, it is likely that several intermediate states exist along the unbinding pathways.

Dynamic force spectroscopy of the digoxigenin–antibody complex

Small ligands and their receptors are widely used non-covalent couplers in various biotech applications. One prominent example, the digoxigenin–antibody complex, was often used to immobilize samples for single molecule force measurements by optical trap or AFM. Here, we employed dynamic AFM spectroscopy to demonstrate that a single digoxigenin–antibody bond is likely to fail even under moderate loading rates. This effect potentially could lower the yield of measurements or even obscure the unbinding data of the sample by the rupture events of the coupler. Immobilization by multiple antibody–antigen bonds, therefore, is highly recommended. The analysis of our data revealed a pronounced loading rate dependence of the rupture force, which we analyzed based on the well-established Bell–Evans-model with two subsequent unbinding barriers. We could show that the first barrier has a width of Δ x 1 = 1.15 nm and a spontaneous rate of k off1 = 0.015 s −1 and the second has a width of Δ x 2 = 0.35 nm and a spontaneous rate of k off2 = 4.56 s −1. In the crossover region between the two regimes, we found a marked discrepancy between the predicted bond rupture probability density and the measured rupture force histograms, which we discuss as non-Markovian contribution to the unbinding process.

Dynamic Mechanism of E2020 Binding to Acetylcholinesterase: A Steered Molecular Dynamics Simulation

The unbinding process of E2020 ((R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methylpiperidine) leaving from the long active site gorge of Torpedo californica acetylcholinesterase (TcAChE) was studied by using steered molecular dynamics (SMD) simulations on a nanosecond scale with different velocities, and unbinding force profiles were obtained. Different from the unbinding of other AChE inhibitors, such as Huperzine A that undergoes the greatest barrier located at the bottleneck of the gorge, the major resistance preventing E2020 from leaving the gorge is from the peripheral anionic site where E2020 interacts intensively with several aromatic residues (e.g., Tyr70, Tyr121, and Trp279) through its benzene ring and forms a strong direct hydrogen bond and a water bridge with Ser286 via its O24. These interactions cause the largest rupture force, approximately 550 pN. It was found that the rotatable bonds of the piperidine ring to the benzene ring and dimethoxyindanone facilitate E2020 to pass the bottleneck through continuous conformation change by rotating those bonds to avoid serious conflict with Tyr121 and Phe330. The aromatic residues lining the gorge wall are the major components contributing to hydrophobic interactions between E2020 and TcAChE. Remarkably, these aromatic residues, acting in three groups as "sender" and "receiver", compose a "conveyer belt" for E2020 entering and leaving the TcAChE gorge.

Change of the unbinding mechanism upon a mutation: A molecular dynamics study of an antibody–hapten complex

We study forced unbinding of fluorescein from the wild type (WT) and a mutant [H(H58)A] of the single-chain variable-fragment (scFv) anti-fluorescein antibody FITC-E2 by molecular dynamics simulations using various pulling techniques. A large number of long simulations were needed to obtain statistically meaningful results as both the wild type and the H(H58)A mutant unbinding occurs through multiple pathways, often with metastable intermediates. For the wild type, the rate-limiting step in the unbinding process corresponds to the breaking of the non-native interactions characteristic of a specific intermediate. The H(H58)A mutation disfavors the occurrence of this intermediate. Two events where the hapten partially unbinds in the absence of pulling force are observed in extensive equilibrium simulations of the wild type, and their analysis indicates that forced unbinding and spontaneous unbinding proceed along similar pathways. The different unbinding mechanisms observed in the simulations suggest a possible reason for the difference in the experimental off-rate between the two antibodies. We predict mutations that are expected to modulate the occurrence of the unbinding intermediate. For two such new mutants [H(H58)A and S(H52)A], our predictions are validated in silico by additional simulations. The accompanying paper in this issue by Honegger et al. reports the X-ray structure of FITC-E2 with a derivative of fluorescein, which was used as the starting conformation for the work presented here.

Force Measurement for Antigen−Antibody Interaction by Atomic Force Microscopy Using a Photograft-Polymer Spacer

To determine the intermolecular force on protein-protein interaction (PPI) by atomic force microscopy (AFM), a photograft-polymer spacer for protein molecules on both surfaces of the substrate and AFM probe tip was developed, and its effectiveness was assessed in a PPI model of a pair of human serum albumin (HSA) and its monoclonal antibody (anti-HSA). A carboxylated photoiniferter, N-(dithiocarboxy)sarcosine, was derivatized on both surfaces of the glass substrate and AFM probe tip, and subsequently water-soluble nonionic vinyl monomers, N,N-dimethylacrylamide (DMAAm), were graft-polymerized on them upon ultraviolet light irradiation. DMAAm-photograft-polymerized spacers with carboxyl groups at the growing chain end but with different chain lengths on both surfaces were prepared. The proteins were covalently bound to the carboxyl terminus of the photograft-polymer chain using a water-soluble condensation agent. The effects of the graft-spacer length on the profile of the force-distance curves and on the unbinding characteristics (unbinding force and unbinding distance) were examined in comparison with those in the case of the commercially available poly(ethylene glycol) (PEG) spacer. The frequency of the nonspecific adhesion force profile was markedly decreased with the use of the photograft spacers. Among the force curves detected, a high frequency of single-peak curves indicating the unbinding process of a single pair of proteins and a very low frequency of multiple-peak profiles were observed for the photograft spacers, regardless of the graft chain length, whereas a high frequency of no-force peaks was noted. These observations were in marked contrast with those for the PEG spacer. The force peak values determined ranged from 88 to 94 pN, irrespective of the type of spacer, while the standard deviation of force distribution observed for the photograft spacer was lower than that for the PEG spacer, indicating that the photograft spacers provide a higher accuracy of force determination.

Directed Binding: A Novel Physical Mechanism That Describes the Directional Motion of Two-Headed Kinesin Motor Proteins

We propose a novel physical mechanism to describe the mode of processive propagation of two-headed kinesin motor proteins along microtubule (MT) filaments. Binding and unbinding of the kinesin heads to and from the MT filament play a crucial role in producing movement. The chemical energy of adenosine triphosphate hydrolysis is used in large part for the unbinding process of kinesin from the MT filament. Importantly, in our model, the binding of each head is to be directionally oriented to the MT filament. Therefore, we treat the two motor domains (heads) as extended objects that are connected with each other by a neck region that contains the kinesin dimerization domain. The head domains recognize tubulin binding sites by feeling the two-dimensional periodic potential from the MT surface and are also subjected to thermal noise. Using experimentally determined results regarding physical parameters of the walk, we develop a simple mathematical and mechanical model in which directed binding of the heads to tubulin results in a directed twist of the molecule, probably in the neck linker region, away from its relaxed state. Unbinding of the head from the filament relaxes the twist and defines the propagation direction. We showed that there must be at least two torsional springs (one for every head) involved that can store elastic energy. Consequently, in our model, it is the internal structure both of the relaxed and tensed-up state and the transition mode between them that define the walking direction of kinesin. We present calculations based on the model that are in good quantitative agreement with experimental observations for kinesin.

Steered molecular dynamics simulations of protein-ligand interactions

Studies of protein-ligand interactions are helpful to elucidating the mechanisms of ligands, providing clues for rational drug design. The currently developed steered molecular dynamics (SMD) is a complementary approach to experimental techniques in investigating the biochemical processes occurring at microsecond or second time scale, thus SMD may provide dynamical and kinetic processes of ligand-receptor binding and unbinding, which cannot be accessed by the experimental methods. In this article, the methodology of SMD is described, and the applications of SMD simulations for obtaining dynamic insights into protein-ligand interactions are illustrated through two of our own examples. One is associated with the simulations of binding and unbinding processes between huperzine A and acetylcholinesterase, and the other is concerned with the unbinding process of α-APAfrom HIV-1 reverse transcriptase.

Water Movement during Ligand Unbinding from Receptor Site

An 1-ns unbinding trajectory of retinol from the bovine serum retinol-binding protein has been obtained from molecular dynamics simulations. The behavior of water during ligand unbinding has never been studied in detail. I described a new method for defining a binding site, located the water molecules involved in the binding site, and examined their movements during unbinding. I found that there were only small changes in the binding site. During unbinding, the number of water molecules inside the binding site decreased, with some water molecules exhibiting movements similar in magnitude to bulk water, and there were rearrangements of the hydrogen bonds. This work represents the first detailed study of the behavior of water during an unbinding process.

Effects of Intermediate Bound States in Dynamic Force Spectroscopy

We revisit some aspects of the interpretation of dynamic force spectroscopy experiments. The standard theory predicts that the typical unbinding force f * is linearly proportional to the logarithm of the loading rate r when a single energy barrier controls the unbinding process. For a more complex situation of N barriers, it predicts at most N linear segments for the f * vs. log( r) curve, each segment characterizing a different barrier. Here we extend this existing picture using a refined approximation, provide a more general analytical formula, and show that in principle up to N( N + 1) / 2 segments can show up experimentally. As a consequence, the determination of the positions and even the number of the energy barriers from the experimental data can be ambiguous. A further possible consequence of a multiple-barrier landscape is a bimodal or multimodal distribution of the unbinding force at certain loading rates, a feature recently observed experimentally.