Stretching single molecules along unbinding and unfolding pathways with the scanning force microscope

Individual molecules can be stretched with a scanning force microscope and the forces required to rupture bonds and to mechanically drive their structures towards new conformations and states can be measured. The tailoring of the experiments, the possibility of carrying them out in quasi-equilibrium conditions, the relationships between single molecule force measurements, and macroscopic kinetics or thermodynamic data are discussed. Mechanochemical experiments are expanding chemistry into new realms between biology and material science.     

Effects of size constraint on water filling process in nanotube

Molecular dynamics (MD) simulation and the potential of mean force (PMF) analysis are used to investigate the structural properties of water molecules near the end of nanotube for the whole process from the initial water filling up to the configuration stabilization inside the carbon nanotubes (CNTs). Numerical simulations showed that when a small-sized nanotube is immersed into the water bath, the size constraint will induce a prevailing orientation for the water molecule to diffuse into the tube and this effect can persist approximately 3.3 angstroms from the end of CNT. As the structure within the CNTs stabilizes, the ambient structural properties can indirectly reflect their corresponding properties inside the nanotube. Our results also showed that there exists a close correlation between the PMF analysis and the results of MD simulations, and the properties at nanometer scale are closely related to the size-constraint effect.     

Mechanically activated rupture of single covalent bonds: evidence of force induced bond hydrolysis

We have used temperature-dependent single molecule force spectroscopy to stretch covalently anchored carboxymethylated amylose (CMA) polymers attached to an amino-functionalized AFM cantilever. Using an Arrhenius kinetics model based on a Morse potential as a one-dimensional representation of covalent bonds, we have extracted kinetic and structural parameters of the bond rupture process. With 35.5 kJ mol(-1), we found a significantly smaller dissociation energy and with 9.0 × 10(2) s(-1) to 3.6 × 10(3) s(-1) also smaller Arrhenius pre-factors than expected for homolytic bond scission. One possible explanation for the severely reduced dissociation energy and Arrhenius pre-factors is the mechanically activated hydrolysis of covalent bonds. Both the carboxylic acid amide and the siloxane bond in the amino-silane surface linker are in principle prone to bond hydrolysis. Scattering, slope and curvature of the scattered data plots indicate that in fact two competing rupture mechanisms are observed.     

Water properties and potential of mean force for hydrophobic interactions of methane and nanoscopic pockets studied by computer simulations

We consider model systems consisting of a methane molecule and hemispherical pockets of subnanometer radii whose walls are made of hydrophobic material. The potential of mean force for process of translocation of the methane molecule from bulk water into the pockets' interior is obtained, based on an explicit solvent molecular dynamics simulations. Accompanying changes in water density around the interacting objects and spatial distribution of solvent's potential energy are analyzed, allowing for interpretation of details of hydrophobic interactions in relation to hydrophobic hydration properties. Applicability of surface area-based models of hydrophobic effect for systems of interest is also investigated. A total work for the translocation process is not dependent on pocket's size, indicating that pocket desolvation has little contribution to free energy changes, which is consistent with the observation that solvent density is significantly reduced inside "unperturbed" pockets. Substantial solvent effects are shown to have a longer range than in case of a well investigated methane pair. A desolvation barrier is present in a smaller pocket system but disappears in the larger one, suggesting that a form of a "hydrophobic collapse" is observed.     

Breaking bonds by mechanical stress: when do electrons decide for the other side?

Using first-principles molecular dynamics, we have simulated reactions that can be induced by mechanical stress in a polymer. We have stretched a small piece of poly(ethylene glycol) (PEG) in water at finite temperature. Both the molecule and the solvent were described quantum mechanically on an equal level. The formation of ions was observed, which corresponds to a heterolytic bond cleavage. We were able to monitor the motion of the electrons during the reactions. Our simulations show that the electron transfer and the breaking of the bond occur almost simultaneously and that both processes are initiated by the approach of a solvent molecule toward the destabilized bond.     

Computer Simulation Study on Adsorption and Conformation of Polymer Chains Driven by External Force

In this work, Monte Carlo simulations are used to study the critical adsorption behaviors of flexible polymer chains under the action of an external driving force F parallel to an attractive flat surface. The critical adsorption temperature T_c decreases linearly with increasing F,indicating that the driving force suppresses the adsorption of polymer. The conformation of polymer is also affected by the driving force.However, the effect of F is dependent on the competition between the driving force and temperature. Under strong force or at low temperature,the polymer is stretched along the direction of the force, while under weak force or at high temperature, the polymer is not stretched. When the force is comparable to the temperature, the polymer may be stretched perpendicular to the driving force, and below T_c, we observe conformational transitions from parallel to perpendicular and again to parallel by decreasing the temperature. We found that the perpendicular stretched conformation leads the polymer chain to synchronously move along the direction of the driving force. Moreover, the conformational transitions are attributed to the competition and cooperation between the driving force and the temperature.     

Mechanism of OMP decarboxylation in orotidine 5'-monophosphate decarboxylase

Despite extensive experimental and theoretical studies, the detailed catalytic mechanism of orotidine 5'-monophosphate decarboxylase (ODCase) remains controversial. In particular simulation studies using high level quantum mechanics have failed to reproduce experimental activation free energy. One common feature of many previous simulations is that there is a water molecule in the vicinity of the leaving CO2 group whose presence was only observed in the inhibitor bound complex of ODCase/BMP. Various roles have even been proposed for this water molecule from the perspective of stabilizing the transition state and/or intermediate state. We hypothesize that this water molecule is not present in the active ODCase/OMP complex. Based on QM/MM minimum free energy path simulations with accurate density functional methods, we show here that in the absence of this water molecule the enzyme functions through a simple direct decarboxylation mechanism. Analysis of the interactions in the active site indicates multiple factors contributing to the catalysis, including the fine-tuned electrostatic environment of the active site and multiple hydrogen-bonding interactions. To understand better the interactions between the enzyme and the inhibitor BMP molecule, simulations were also carried out to determine the binding free energy of this special water molecule in the ODCase/BMP complex. The results indicate that the water molecule in the active site plays a significant role in the binding of BMP by contributing approximately -3 kcal/mol to the binding free energy of the complex. Therefore, the complex of BMP plus a water molecule, instead of the BMP molecule alone, better represents the tight binding transition state analogue of ODCase. Our simulation results support the direct decarboxylation mechanism and highlight the importance of proper recognition of protein bound water molecules in the protein-ligand binding and the enzyme catalysis.     

Functionalized self-assembled monolayers for measuring single molecule lectin carbohydrate interactions

The specific interactions between sugar-binding proteins (lectins) and their complementary carbohydrates mediate several complex biological functions. There is a great deal of interest in uncovering the molecular basis of these interactions. In this study, we demonstrate the use of an efficient one-step amination reaction strategy to fabricate carbohydrate arrays based on mixed self-assembled monolayers. These allow specific lectin carbohydrate interactions to be interrogated at the single molecule level via AFM. The force required to directly rupture the multivalent bonds between Concanavalin A (Con A) and mannose were subsequently determined by chemical force microscopy. The mixed self-assembled monolayer provides a versatile platform with active groups to attach a 1-amino-1-deoxy sugar or a protein (Con A) while minimizing non-specific adhesion enabling quick and reliable detection of rupture forces. By altering the pH of the environment, the aggregation state of Con A was regulated, resulting in different dominant rupture forces, corresponding to di-, tri- and multiple unbinding events. We estimate the value of the rupture force for a single Con A-mannose bond to be 95 ± 10 pN. The rupture force is consistent even when the positions of the binding molecules are switched. We show that this synthesis strategy in conjunction with a mixed SAM allows determination of single molecules bond with high specificity, and may be used to investigate lectin carbohydrate interactions in the form of carbohydrate arrays as well as lectin arrays.     

Effects of hydrogen-bond environment on single particle and pair dynamics in liquid water

We have performed molecular dynamics simulations of liquid water at 298 and 258 K to investigate the effects of hydrogen-bond environment on various single-particle and pair dynamical properties of water molecules at ambient and supercooled conditions. The water molecules are modelled by the extended simple point charge (SPC/E) model. We first calculate the distribution of hydrogen-bond environment in liquid water at both temperatures and then investigate how the self-diffusion and orientational relaxation of a single water molecule and also the relative diffusion and relaxation of the hydrogen-bond of a water pair depend on the nature of the hydrogen-bond environment of the tagged molecules. We find that the various dynamical quantities depend significantly on the hydrogen-bond environment, especially at the supercooled temperature. The present study provides a molecular-level insight into the dynamics of liquid water under ambient and supercooled conditions.     

Effect of temperature and water content on the shear viscosity of the ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide as studied by atomistic simulations

Atomistic simulations are conducted to examine the dependence of the viscosity of 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide on temperature and water content. A nonequilibrium molecular dynamics procedure is utilized along with an established fixed charge force field. It is found that the simulations quantitatively capture the temperature dependence of the viscosity as well as the drop in viscosity that occurs with increasing water content. Using mixture viscosity models, we show that the relative drop in viscosity with water content is actually less than that that would be predicted for an ideal system. This finding is at odds with the popular notion that small amounts of water cause an unusually large drop in the viscosity of ionic liquids. The simulations suggest that, due to preferential association of water with anions and the formation of water clusters, the excess molar volume is negative. This means that dissolved water is actually less effective at lowering the viscosity of these mixtures when compared to a solute obeying ideal mixing behavior. The use of a nonequilibrium simulation technique enables diffusive behavior to be observed on the time scale of the simulations, and standard equilibrium molecular dynamics resulted in sub-diffusive behavior even over 2 ns of simulation time.     

Molecular basis for dimethylsulfoxide (DMSO) action on lipid membranes

Dimethylsulfoxide (DMSO) is an aprotic solvent that has the ability to induce cell fusion and cell differentiation and enhance the permeability of lipid membranes. It is also an effective cryoprotectant. Insights into how this molecule modulates membrane structure and function would be invaluable toward regulating the above processes and for developing chemical means for enhancing or hindering the absorption of biologically active molecules, in particular into or via the skin. We show here by means of molecular simulations that DMSO can induce water pores in dipalmitoyl-phosphatidylcholine bilayers and propose this to be a possible pathway for the enhancement of penetration of actives through lipid membranes. DMSO also causes the membrane to become floppier, which would enhance permeability, facilitate membrane fusion, and enable the cell membrane to accommodate osmotic and mechanical stresses during cryopreservation.     

Transient violations of the second law of thermodynamics in protein unfolding examined using synthetic atomic force microscopy and the fluctuation theorem

The synthetic atomic force microscopy (AFM) method is developed to simulate a periodically replicated atomistic system subject to force and length fluctuations characteristic of an AFM experiment. This new method is used to examine the forced-extension and subsequent rupture of the alpha-helical linker connecting periodic images of a spectrin protein repeat unit. A two-dimensional potential of mean force (PMF) along the length and a reaction coordinate describing the state of the linker was calculated. This PMF reveals that the basic material properties of the spectrin repeat unit are sensitive to the state of linker, an important feature that cannot be accounted for in a one-dimensional PMF. Furthermore, nonequilibrium simulations were generated to examine the rupture event in the context of the fluctuation theorem. These atomistic simulations demonstrate that trajectories which are in apparent violation of the second law can overcome unfolding barriers at significantly reduced rupture forces.     

An implemented potential of non-rigid water molecules for molecular dynamics simulations

The central force model for a water molecule is corrected by adding a three-body term. The present potential fits both an accurate ab initio potential energy surface and the fundamental vibrational frequencies of gas-phase water. The three-body terms allow us to reproduce the gas-phase IR spectrum by molecular dynamics simulations. Some problems connected with MD simulations of IR spectra are discussed.     

Volume of a nanoscale water bridge

Water bridges formed through capillary condensation at nanoscale contacts first stretch and then break during contact rupture. Atomic force microscopy (AFM) pull-off experiments performed in air with hydrophilic tips and samples show that stretched nanoscopic water bridges are in mechanical equilibrium with the external pull-off force acting at the contact but not in thermodynamic equilibrium with the water vapor in air. The experimental findings are explained by a theoretical model that considers constant water volume and decrease of water meniscus curvature during meniscus stretching. The model predicts that the water bridge breakup distance will be roughly equal to the cubic root of the water bridge volume. A thermodynamic instability was noticed for large water bridges formed at the contact of a blunt AFM tip (curvature radius of 400 nm) with a flat sample. In this case, experiments showed rise and stabilization of the volume of the water at the contact in about 1 s. For sharp AFM tips (curvature radius below 50 nm), the experiments indicated that formation of stable water bridges occurs in a much shorter time (below 5 ms).     

Understanding solvent effects in the selective conversion of fructose to 5-hydroxymethyl-furfural: a molecular dynamics investigation

Selective conversion of fructose to 5-hydroxymethyl-furfural (HMF) involves the participation of high-boiling solvents like dimethyl sulfoxide (DMSO). In order to replace DMSO with low-boiling solvents, it is imperative that we understand the effect of DMSO solvation in protecting (i) HMF from rehydration and humins formation reactions and (ii) fructose from side reactions, other than its dehydration to HMF. In the present work, molecular dynamics simulations of HMF and fructose in water and in water-DMSO mixtures are carried out using the OPLS-AA force field. Radial pair distribution functions, coordination numbers and the hydrogen-bond network between the HMF/fructose molecule and the solvent molecules are analysed. The local 3-dimensional picture of the arrangement of solvent molecules around the solute, which cannot be accessed from pair distribution functions, is also computed. We show preferential coordination of DMSO around HMF and explain how this could provide a shielding effect to the HMF molecule, thus protecting it from further rehydration to levulinic acid and formic acid and from humins formation. In the case of fructose, the presence of DMSO also reduces the number of water molecules in the immediate vicinity of fructose. Though fewer water molecules coordinate around fructose, they are bound strongly to it. Analysis of the local 3-dimensional arrangement of DMSO molecules suggests that it protects the fructose molecule from side reactions that would lead to condensation or reversion products. However, the presence of DMSO molecules does not hamper the water molecules coming into contact with the oxygen atom of the hydroxyl groups of fructose, which is required for a proton transfer from water to fructose, to initiate the dehydration reaction to HMF.     

A force field for monosaccharides and (1 → 4) linked polysaccharides

A force field for monosaccharides that can be extended to (1 → 4) linked polysaccharides has been developed for the AMBER potential function. The resulting force field is consistent with the existing AMBER force field for proteins and nucleic acids. Modifications to the standard AMBER OH force constant and to the Lennard-Jones parameters were made. Furthermore, a 10–12 nonbonded term was included between the hydroxyl hydrogen of the saccharide and the water oxygen (TIP3P, SPC/E, etc.) to reproduce better the water–saccharide intermolecular distances. STO-3G electrostatic potential (ESP) charges were used to represent the electrostatic interactions between the saccharide and its surrounding environment. To obtain charges for polysaccharides, a scheme was developed to piece together saccharide residues through 1 → 4 connections while still retaining a net neutral charge on the molecule as a whole. Free energy perturbation (FEP) simulations of D -glucose and D -mannose in water were performed to test the resulting force field. The FEP simulations demonstrate that AMBER overestimates intramolecular interaction energies, suggesting that further improvements are needed in this part of the force field. To test further the reliability of the parameters, a molecular dynamics (MD) simulation of α-D -glucose in water was also performed. The MD simulation was able to produce structural and conformational results that are in accord with experimental evidence and previous theoretical results. Finally, a relaxed conformational map of β-maltose was assembled and it was found that the present force field is consistent with available theoretical and experimental results. © 1994 by John Wiley & Sons, Inc.     

Nonlinearly additive forces in multivalent ligand binding to a single protein revealed with force spectroscopy

We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between concanavalin A (ConA) and alpha-D-mannose but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, they do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 68, and 85 pN at a loading rate of 650 +/- 25 pN/s, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and nonspecific binding. We analyze the binding configuration (i.e., serial vs parallel connections) through fitting the polymer stretching data with modified wormlike chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously.     

Modelling a nematic liquid crystal

The bulk phase liquid crystalline behaviour of a cyclic siloxane with a pentamethylcyclosiloxane core and biphenyl-4-allyloxybenzoate mesogens (BCS) was studied using molecular dynamics (MD) and wide angle X-ray analysis. This material exhibits partial crystallinity at room temperature and liquid crystalline behaviour above 120° C. For the MD simulations an ensemble of 27 molecules with 135 mesogenic units was simulated and a molecular mechanics force field was used to model the structural anisotropy of the siloxane molecules. Simulations were carried out both at room temperature and at an elevated temperature (425 K). Room temperature simulations showed that, contrary to our initial assumptions, the low energy molecular conformations were not cylindrical but splayed in shape. During the simulation a smectic-like, tilted layer structure was found to evolve for the cluster when full atom potentials were used, while no such development was observed when electrostatic interactions were neglected. The presence of a tilted layered structure was also suggested by the X-ray data. These results indicate that long range electrostatic interactions are significant for the molecular system under study. In order to calculate the orientational order parameter, the orientation of the molecular axis had to be determined. This was achieved by describing the mesogen shapes to be ellipsoidal and defining the principal axis of the ellipsoids to be the molecular directors. By sampling over 200 ps of simulation at 425 K, the time averaged order parameter (S) was calculated. The calculated S of 0.36 was comparable to the value of 0.4-0.45 found from the experimental data. Apart from providing insight into the relative importance of the various competing forces in the formation of the liquid crystalline phase, these simulations are also expected to be useful in predicting the mesophase behaviour of liquid crystalline systems.     

Stretching of hydrogen-bonded OH in the lowest singlet excited electronic state of water dimer

The lowest singlet excited electronic state of water monomer in the gas phase is strictly dissociative along a OH stretch coordinate but changes its nature when the stretched OH moiety is hydrogen bonded to a neighboring water molecule. This work extends previous exploration of the water dimer excited singlet potential-energy surface, using computational methods that are reliable even at geometries well removed from the ground-state equilibrium. First, the hydrogen-bonded OH moiety is stretched far enough to establish the existence of a barrier that is sufficient to support a quasibound vibrational state of the OH oscillator near the Franck-Condon region. Second, the constraint of an icelike structure is relaxed, and it is found that a substantial fraction of liquidlike structures also supports a quasibound vibrational state. These potential-energy explorations on stretching of the hydrogen-bonded OH moiety in a water dimer are discussed as a model for understanding the initial dynamics upon excitation into the lowest excited singlet state of condensed water. The possibility is raised that the excited-state lifetime may be long enough to allow for exciton migration, which would provide a mechanism for energy transport in condensed water phases.     

Binding of hydrogen bonding solutes at liquid–vapour interfaces of molecular fluids

We have calculated the potential of mean force (PMF) for the transfer of a solute molecule across a liquid–vapour interface for four different systems: (a) one methanol molecule in water, (b) one water molecule in methanol, (c) one acetonitrile molecule in water and (d) one water molecule in acetonitrile by means of constrained molecular dynamics simulations. A minimum of the PMF is found near the Gibbs dividing surface for methanol and acetonitrile solutes although the degree of surface activity is found to be somewhat different due, in part, to varying hydrogen bonding nature of these two solutes.