Rebinding Events Research Articles

Influence of electronic polarization on the binding of anions to a chloride-pumping rhodopsin

The functional properties of some biological ion channels and membrane transport proteins are proposed to exploit anion-hydrophobic interactions. Here, we investigate a chloride-pumping rhodopsin as an example of a membrane protein known to contain a defined anion binding site composed predominantly of hydrophobic residues. Using molecular dynamics simulations, we explore Clˉ binding to this hydrophobic site and compare the dynamics arising when electronic polarization is neglected (CHARMM36 [c36] fixed-charge force field), included implicitly (via the prosECCo force field), or included explicitly (through the polarizable force field, AMOEBA). Free energy landscapes of Clˉ moving out of the binding site and into bulk solution demonstrate that the inclusion of polarization results in stronger ion binding and a second metastable binding site in chloride-pumping rhodopsin. Simulations focused on this hydrophobic binding site also indicate longer binding durations and closer ion proximity when polarization is included. Furthermore, simulations reveal that Clˉ within this binding site interacts with an adjacent loop to facilitate rebinding events that are not observed when polarization is neglected. These results demonstrate how the inclusion of polarization can influence the behavior of anions within protein binding sites and can yield results comparable with more accurate and computationally demanding methods.

Influence of electronic polarization in a chloride-pumping rhodopsin binding site.

Biological ion channels and membrane transport proteins are suggested to utilize anion-hydrophobic interactions to achieve selective anion transport. Here, we investigate a chloride-pumping rhodopsin (ClR) as an example of a Clˉ-selective protein known to contain a defined binding site composed predominantly of hydrophobic residues. Using molecular dynamics (MD) simulations, we explore Clˉ binding to this hydrophobic binding site and compare the dynamics arising when electronic polarization is either (i) neglected (CHARMM36 (c36) fixed-charge force field), (ii) included implicitly (via the prosECCo force field based on the electronic-continuum-correction method), or (iii) included explicitly (through the polarizable force field, AMOEBA). Free energy landscapes of a Clˉ moving out of the binding site and into bulk solution demonstrate stronger ion binding and exhibit a second metastable site with the inclusion of polarization effects. Simulations of a small protein fragment containing the binding site indicate longer binding durations and closer ion proximity with the inclusion of polarization. Finally, examination of larger protein fragment simulations consisting of the binding site and a neighboring loop reveal a mechanism that facilitates Clˉ rebinding events which are not observed with the non-polarizable force field. These results demonstrate the importance of modelling polarization (effects) which can influence the behavior of anions within real protein binding sites and suggest important mechanistic insights.

Robustness of DNA looping across multiple cell divisions in individual bacteria

DNA looping has emerged as a central paradigm of transcriptional regulation, as it is shared across many living systems. One core property of DNA looping-based regulation is its ability to greatly enhance repression or activation of genes with only a few copies of transcriptional regulators. However, this property based on a small number of proteins raises the question of the robustness of such a mechanism with respect to the large intracellular perturbations taking place during growth and division of the cell. Here we address the issue of sensitivity to variations of intracellular parameters of gene regulation by DNA looping. We use the lac system as a prototype to experimentally identify the key features of the robustness of DNA looping in growing Escherichia coli cells. Surprisingly, we observe time intervals of tight repression spanning across division events, which can sometimes exceed 10 generations. Remarkably, the distribution of such long time intervals exhibits memoryless statistics that is mostly insensitive to repressor concentration, cell division events, and the number of distinct loops accessible to the system. By contrast, gene regulation becomes highly sensitive to these perturbations when DNA looping is absent. Using stochastic simulations, we propose that the observed robustness to division emerges from the competition between fast, multiple rebinding events of repressors and slow initiation rate of the RNA polymerase. We argue that fast rebinding events are a direct consequence of DNA looping that ensures robust gene repression across a range of intracellular perturbations.

Elasticity-associated rebinding rate of molecular bonds between soft elastic media

A quantitative understanding of how cells interact with their extracellular matrix via molecular bonds is fundamental for many important processes in cell biology and engineering. In these interactions, the deformability of cells and matrix are usually comparable with that of the bonds, making their rebinding events globally coupled with the deformation states of whole systems. Unfortunately, this important principle is not realized or adopted in most conventional theoretical models for analyzing cellular adhesions. In this study, we considered a new theoretical model of a cluster of ligand-receptor bonds between two soft elastic bodies, in which the rebinding rates of ligands to receptors are described, by considering the deformation of the overall system under the influence of bond distributions. On the basis of theory of continuum and statistical mechanics, we obtained an elasticity-associated rebinding rate of open bonds in a closed analytical form that highly depends on the binding states and distributions of all other bonds as well as on the overall deformation energy stored in the elastic bodies and all closed bonds. On the basis of this elasticity-associated rebinding rate and by performing Monte Carlo simulations, we uncovered new mechanisms underlying the adhesion stability of molecular-bond clusters associated with deformable elastic bodies. Moreover, we revealed that the rebinding processes of molecular bonds is not only dependent on interfacial separation but is related to overall energy. This newly proposed rebinding rate may substantially improve our understanding of how cells adapt to their microenvironments by adjusting their mechanical properties through cytoskeleton remodeling.

Protein-Protein Interaction-Gaussian Accelerated Molecular Dynamics (PPI-GaMD): Characterization of Protein Binding Thermodynamics and Kinetics.

Protein-protein interactions (PPIs) play key roles in many fundamental biological processes such as cellular signaling and immune responses. However, it has proven challenging to simulate repetitive protein association and dissociation in order to calculate binding free energies and kinetics of PPIs due to long biological timescales and complex protein dynamics. To address this challenge, we have developed a new computational approach to all-atom simulations of PPIs based on a robust Gaussian accelerated molecular dynamics (GaMD) technique. The method, termed "PPI-GaMD", selectively boosts interaction potential energy between protein partners to facilitate their slow dissociation. Meanwhile, another boost potential is applied to the remaining potential energy of the entire system to effectively model the protein's flexibility and rebinding. PPI-GaMD has been demonstrated on a model system of the ribonuclease barnase interactions with its inhibitor barstar. Six independent 2 μs PPI-GaMD simulations have captured repetitive barstar dissociation and rebinding events, which enable calculations of the protein binding thermodynamics and kinetics simultaneously. The calculated binding free energies and kinetic rate constants agree well with the experimental data. Furthermore, PPI-GaMD simulations have provided mechanistic insights into barstar binding to barnase, which involves long-range electrostatic interactions and multiple binding pathways, being consistent with previous experimental and computational findings of this model system. In summary, PPI-GaMD provides a highly efficient and easy-to-use approach for binding free energy and kinetics calculations of PPIs.

The Pleckstrin Homology Domain of PLCδ1 Exhibits Complex Dissociation Properties at the Inner Leaflet of Plasma Membrane Sheets.

Using total internal reflection fluorescence microscopy, we followed the dissociation of GFP-tagged pleckstrin homology (PH) domains of AKT and PLCδ1 from the plasma membranes of rapidly unroofed cells. We found that the AKT-PH-GFP and PLCδ1-PH-GFP dissociation kinetics can be distinguished by their effective koff values of 0.39 ± 0.05 and 0.56 ± 0.16 s-1, respectively. Furthermore, we identified substantial rebinding events in measurements of PLCδ1-PH-GFP dissociation kinetics. By applying inositol triphosphate (IP3) to samples during the unroofing process, we measured a much larger koff of 1.54 ± 0.42 s-1 for PLCδ1-PH-GFP, indicating that rebinding events are significantly suppressed through competitive action by IP3 for the same PH domain binding site as phosphatidylinositol 4,5-bisphosphate (PIP2). We discuss the complex character of our PLCδ1-PH-GFP fluorescence decays in the context of membrane receptor and ligand theory to address the question of how free PIP2 levels modulate the interaction between membrane-associated proteins and the plasma membrane.

Influence of molecular rebinding on the reaction rate of complex formation.

We simulated Brownian diffusion and reaction-diffusion processes to study the influence of molecular rebinding on the reaction rates of bimolecular reactions. We found that the number of rebinding events, Nreb, is proportional to the target's size and inversely proportional to the diffusion coefficient D and simulation time-step Δt. We found the proportionality constant close to π-1/2. We confirmed that Nreb is defined as a ratio of the activation-limited rate constant ka to the diffusion-limited rate constant, kD. We provide the formula describing the reactivity coefficient κ, modelling the transient-native complex transition for the activation-controlled reaction rates. We show that κ is proportional to (D/Δt)1/2. Finally, we apply our rebinding-including reaction rate model to the real reactions of photoacid dissociation and protein association. Based on literature data for both types of reactions, we found the Δt time-scale. We show that for the photodissociation of a proton, the Δt is equal to 171 ± 18 fs and the average number of rebinding events is approximately equal to 40. For proteins, Δt is of the order of 100 ps with around 20 rebinding events. In both cases the timescale is similar to the timescale of fluctuation of the solvent molecules surrounding the reactants; vibrations and bending in the case of photoacid dissociation and diffusional motion for proteins.

Receptor-Ligand Rebinding Kinetics in Confinement

Rebinding kinetics of molecular ligands plays a key role in the operation of biomachinery, from regulatory networks to protein transcription, and is also a key factor in design of drugs and high-precision biosensors. In this study, we investigate initial release and rebinding of ligands to their binding sites grafted on a planar surface, a situation commonly observed in single-molecule experiments and that occurs in vivo, e.g., during exocytosis. Via scaling arguments and molecular dynamic simulations, we analyze the dependence of nonequilibrium rebinding kinetics on two intrinsic length scales: the average separation distance between the binding sites and the total diffusible volume (i.e., height of the experimental reservoir in which diffusion takes place or average distance between receptor-bearing surfaces). We obtain time-dependent scaling laws for on rates and for the cumulative number of rebinding events. For diffusion-limited binding, the (rebinding) on rate decreases with time via multiple power-law regimes before the terminal steady-state (constant on-rate) regime. At intermediate times, when particle density has not yet become uniform throughout the diffusible volume, the cumulative number of rebindings exhibits a novel, to our knowledge, plateau behavior because of the three-dimensional escape process of ligands from binding sites. The duration of the plateau regime depends on the average separation distance between binding sites. After the three-dimensional diffusive escape process, a one-dimensional diffusive regime describes on rates. In the reaction-limited scenario, ligands with higher affinity to their binding sites (e.g., longer residence times) delay entry to the power-law regimes. Our results will be useful for extracting hidden timescales in experiments such as kinetic rate measurements for ligand-receptor interactions in microchannels, as well as for cell signaling via diffusing molecules.

Unbiased Atomistic Insight into the Mechanisms and Solvent Role for Globular Protein Dimer Dissociation.

Association and dissociation of proteins are fundamental processes in nature. Although simple to understand conceptually, the details of the underlying mechanisms and role of the solvent are poorly understood. Here, we investigate the dissociation of the hydrophilic β-lactoglobulin dimer by employing transition path sampling. Analysis of the sampled path ensembles reveals a variety of mechanisms: (1) a direct aligned dissociation (2) a hopping and rebinding transition followed by unbinding, and (3) a sliding transition before unbinding. Reaction coordinate and transition-state analysis predicts that, besides native contact and neighboring salt-bridge interactions, solvent degrees of freedom play an important role in the dissociation process. Bridging waters, hydrogen-bonded to both proteins, support contacts in the native state and nearby lying transition-state regions, whereas they exhibit faster dynamics in further lying transition-state regions, rendering the proteins more mobile and assisting in rebinding. Analysis of the structure and dynamics of the solvent molecules reveals that the dry native interface induces enhanced populations of both disordered hydration water near hydrophilic residues and tetrahedrally ordered hydration water nearby hydrophobic residues. Although not exhaustive, our sampling of rare unbiased reactive molecular dynamics trajectories enhances the understanding of protein dissociation via complex pathways including (multiple) rebinding events.

Free Energy of Binding and Mechanism of Interaction for the MEEVD-TPR2A Peptide-Protein Complex.

The association between the MEEVD C-terminal peptide from the heat shock protein 90 (Hsp90) and tetratricopeptide repeat A (TPR2A) domain of the heat shock organizing protein (Hop) is a useful prototype to study the fundamental molecular details about the Hop-Hsp90 interaction. We study here the mechanism of binding/unbinding and compute the standard binding free energy and potential of mean force for the association of the MEEVD peptide to the TPR2A domain using the Adaptive Biasing Force (ABF) methodology. We observe conformational changes of the peptide and the protein receptor induced by binding. We measure the binding free energy of -8.4 kcal/mol, which is consistent with experimental estimates. The simulations achieve multiple unbinding and rebinding events along a consistent pathway connecting the binding site to solvent. The MEEVD peptide slowly dissociates disrupting the hydrogen bonds first, then tilting on the side while preserving the interaction with the side chain of residue Asp 5 of the peptide. After this initial displacement, the peptide completely dissociates and moves into the solvent. Rebinding of the MEEVD peptide from the solvent to the receptor binding site occurs slowly through the portal of entry. Unbinding and rebinding go through intermediate states characterized by the peptide interacting with a lateral helix, helix A1, of the receptor with mainly Asp 5, Val 4, and Glu 3 of the peptide. This newly discovered intermediate structure is characterized by numerous contacts with the receptor which lead to complete formation of the bound complex. The structure of the bound complex obtained after rebinding is structurally very similar to the crystal structure of the complex (0.48 Å root-mean square deviation). The residues Asp 5, Val 4, and Glu 3 adopt conformations and intermolecular contacts with excellent structural similarity with the native ones. Finally, the dissociation and reassociation of MEEVD induce hydration/dehydration transitions, which provide insights on the role of desolvation and solvation processes in protein-peptide binding.

Ligand–receptor binding kinetics in surface plasmon resonance cells: a Monte Carlo analysis

Surface plasmon resonance (SPR) chips are widely used to measure association and dissociation rates for the binding kinetics between two species of chemicals, e.g., cell receptors and ligands. It is commonly assumed that ligands are spatially well mixed in the SPR region, and hence a mean-field rate equation description is appropriate. This approximation however ignores the spatial fluctuations as well as temporal correlations induced by multiple local rebinding events, which become prominent for slow diffusion rates and high binding affinities. We report detailed Monte Carlo simulations of ligand binding kinetics in an SPR cell subject to laminar flow. We extract the binding and dissociation rates by means of the techniques frequently employed in experimental analysis that are motivated by the mean-field approximation. We find major discrepancies in a wide parameter regime between the thus extracted rates and the known input simulation values. These results underscore the crucial quantitative importance of spatio-temporal correlations in binary reaction kinetics in SPR cell geometries, and demonstrate the failure of a mean-field analysis of SPR cells in the regime of high Damköhler number , where the spatio-temporal correlations due to diffusive transport and ligand–receptor rebinding events dominate the dynamics of SPR systems.

DNA binding proteins explore multiple local configurations during docking via rapid rebinding.

Finding the target site and associating in a specific orientation are essential tasks for DNA-binding proteins. In order to make the target search process as efficient as possible, proteins should not only rapidly diffuse to the target site but also dynamically explore multiple local configurations before diffusing away. Protein flipping is an example of this second process that has been observed previously, but the underlying mechanism of flipping remains unclear. Here, we probed the mechanism of protein flipping at the single molecule level, using HIV-1 reverse transcriptase (RT) as a model system. In order to test the effects of long-range attractive forces on flipping efficiency, we varied the salt concentration and macromolecular crowding conditions. As expected, increased salt concentrations weaken the binding of RT to DNA while increased crowding strengthens the binding. Moreover, when we analyzed the flipping kinetics, i.e. the rate and probability of flipping, at each condition we found that flipping was more efficient when RT bound more strongly. Our data are consistent with a view that DNA bound proteins undergo multiple rapid re-binding events, or short hops, that allow the protein to explore other configurations without completely dissociating from the DNA.

Investigation of ion binding in chlorite dismutases by means of molecular dynamics simulations.

Chlorite dismutases are prokaryotic heme b oxidoreductases that convert chlorite to chloride and dioxygen. It has been postulated that during turnover hypochlorite is formed transiently, which might be responsible for the observed irreversible inactivation of these iron proteins. The only charged distal residue in the heme cavity is a conserved and mobile arginine, but its role in catalysis and inactivation is not fully understood. In the present study, the pentameric chlorite dismutase (Cld) from the bacterium Candidatus Nitrospira defluvii was probed for binding of the low spin ligand cyanide, the substrate chlorite, and the intermediate hypochlorite. Simulations were performed with the enzyme in the ferrous, ferric, and compound I state. Additionally, the variant R173A was studied. We report the parametrization for the GROMOS force field of the anions ClO–, ClO2–, ClO3–, and ClO4– and describe spontaneous binding, unbinding, and rebinding events of chlorite and hypochlorite, as well as the dynamics of the conformations of Arg173 during simulations. The findings suggest that (i) chlorite binding to ferric NdCld occurs spontaneously and (ii) that Arg173 is important for recognition and to impair hypochlorite leakage from the reaction sphere. The simulation data is discussed in comparison with experimental data on catalysis and inhibition of chlorite dismutase.

A Predicted Binding Site for Cholesterol on the GABAA Receptor

Modulation of the GABA type A receptor (GABAAR) function by cholesterol and other steroids is documented at the functional level, yet its structural basis is largely unknown. Current data on structurally related modulators suggest that cholesterol binds to subunit interfaces between transmembrane domains of the GABAAR. We construct homology models of a human GABAAR based on the structure of the glutamate-gated chloride channel GluCl of Caenorhabditis elegans. The models show the possibility of previously unreported disulfide bridges linking the M1 and M3 transmembrane helices in the α and γ subunits. We discuss the biological relevance of such disulfide bridges. Using our models, we investigate cholesterol binding to intersubunit cavities of the GABAAR transmembrane domain. We find that very similar binding modes are predicted independently by three approaches: analogy with ivermectin in the GluCl crystal structure, automated docking by AutoDock, and spontaneous rebinding events in unbiased molecular dynamics simulations. Taken together, the models and atomistic simulations suggest a somewhat flexible binding mode, with several possible orientations. Finally, we explore the possibility that cholesterol promotes pore opening through a wedge mechanism.

How Accurately Can a Single Receptor Measure Ligand Concentrations?

Biological sensory systems often display remarkable accuracy and sensitivity, sometimes detecting the presence of just a few molecules in the environment (1). During embryo growth, important developmental decisions are sometimes made based on the positional information in just a few molecules (2). Therefore, biophysicists have wondered what determines the fundamental precision limit of biological sensory performance. A new study by Kaizu et al. (3) revisits this important problem and demonstrates that statistical fluctuations and many-body correlations both can influence this fundamental sensory limit. Their result represents an improved estimate of the sensory precision limit based on fundamental physics. The elementary event in sensory processes is the binding of a diffusing ligand molecule to a single sensory receptor. In what is now a classic in the field, Berg and Purcell (4) considered this problem in the regime where the binding reaction is diffusion-limited and receptor switching is Markovian. In this case the receptor switches between bound and unbound states with exponentially distributed waiting times. They obtained that the precision of ligand concentration measured by the receptor is δcc=24Dσc(1−n¯)T, (1) where c is the ligand concentration in the environment and δc is the error in the concentration estimate. The value T is the time interval of measurement. The value σ is the receptor size and n¯ is average probability of finding the receptor bound with ligand. This result makes intuitive sense (5), but because it is in the diffusion-limited regime, the ligand binds immediately when it reaches the receptor and reaction rate constants do not appear explicitly. The applicability of this result outside of the diffusion-limit regime is not clear. Bialek and Setayeshgar (5) sought to generalize the result from Berg and Purcell (4) by considering the binding-unbinding dynamics together with the ligand diffusion process. They utilized the fluctuation-dissipation theorem (FDT) to solve the coupled diffusion-reaction problem. FDT allows one to estimate the time it takes for a fluctuation in a system to relax to the equilibrium average. In our case, the fluctuation in consideration is the binding occupancy of the receptor. Bialek and Setayeshgar (5) predicted that the ligand concentration measurement error is related to fluctuation of the receptor occupancy around the mean, and the result from Berg and Purcell (4) should be modified to δcc=1πDσcT+2kac(1−n¯)T, (2) where ka is the receptor associate rate, given that the receptor and ligand are at contact. This result incorporates important spatial correlation effects from diffusion. However, in the diffusion-limited regime, the second term in the square-root should disappear and only diffusion effects remain. In this limit, Eq. 2 does not agree with the result from Berg and Purcell (4), apart from a geometric factor of 2π. Indeed, when the average receptor occupancy is high (n¯≈1), Eq. 2 does not diverge as in Eq. 1. But when the receptor is constantly occupied, it cannot measure the concentration field and the error should diverge. Kaizu et al. (3) revisited this problem. Their analysis resolves the conflicting results on fundamental limits to the precision in chemical sensing. Kaizu et al. (3) argues that the concentration measurement limit should be δcc=12πDσc(1−n¯)T+2kac(1−n¯)T. (3) They then validate this result by performing rigorous simulation of the reaction-diffusion process. In this new result, while the second reaction term agrees with Bialek and Setayeshgar (5), the diffusion term agrees with Berg and Purcell (4). Kaizu et al. (3) speculates that linearization from FDT misses some ligand rebinding events and receptor-ligand correlations. These nonlinear effects may be the reason behind the apparent discrepancy between Eqs. 1 and 2. An improved estimate of concentration measurement limit has implications in all sorts of biological sensory systems, ranging from chemotaxis in eukaryotes and prokaryotes, to the sense of smell. Other applications include developmental systems where tissue patterning requires sensitive measurement of spatial morphogen concentrations. Perhaps morphological variations can be ultimately traced to slight errors in concentration measurements. The work of Kaizu et al. (3) completes a piece of the puzzle in this important problem. However, it only treats the case of single isolated receptors. Cells can employ multiple receptors and utilize energy to accurately detect signals at a wide range of ligand concentrations (6). Receptor-receptor correlations and their implication in sensory systems are also interesting. Physical effects and statistical fluctuations are sure to play roles in complex receptor arrays as well.

Analysis and Assay of Oseltamivir-Resistant Mutants of Influenza Neuraminidase via Direct Observation of Drug Unbinding and Rebinding in Simulation

The emergence of influenza drug resistance is a major public health concern. The molecular basis of resistance to oseltamivir (Tamiflu) is investigated using a computational assay involving multiple 500 ns unrestrained molecular dynamics (MD) simulations of oseltamivir complexed with mutants of H1N1-2009 influenza neuraminidase. The simulations, accelerated using graphics processors (GPUs), and using a fully explicit model of water, are of sufficient length to observe multiple drug unbinding and rebinding events. Drug unbinding occurs during simulations of known oseltamivir-resistant mutants of neuraminidase. Molecular-level rationalizations of drug resistance are revealed by analysis of these unbinding trajectories, with particular emphasis on the dynamics of the mutant residues. The results indicate that MD simulations can predict weakening of binding associated with drug resistance. In addition, visualization and analysis of binding site water molecules reveal their importance in stabilizing the binding mode of the drug. Drug unbinding is accompanied by conformational changes, driven by the mutant residues, which results in flooding of a key pocket containing tightly bound water molecules. This displaces oseltamivir, allowing the tightly bound water molecules to be released into bulk. In addition to the role of water, analysis of the trajectories reveals novel behavior of the structurally important 150-loop. Motion of the loop, which can move between an open and closed conformation, is intimately associated with drug unbinding and rebinding. Opening of the loop occurs coincidentally with drug unbinding, and interactions between oseltamivir and the loop seem to aid in the repositioning of the drug back into an approximation of its original binding mode on rebinding. The similarity of oseltamivir to a transition state analogue for neuraminidase suggests that the dynamics of the loop could play an important functional role in the enzyme, with loop closing aiding in binding of the substrate and loop opening aiding the release of the product.

Modulation of ligand–heme reactivity by binding pocket residues demonstrated in cytochrome c' over the femtosecond–second temporal range

The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affinity for their chosen ligand, is fundamental to the existence of life. These processes are often controlled by precise structural arrangements in proteins, with heme pocket residues driving reactivity and specificity. One such protein is cytochrome c', which has the ability to bind nitric oxide (NO) and carbon monoxide (CO) on opposite faces of the heme, a property that is shared with soluble guanylate cycle. Like soluble guanylate cyclase, cytochrome c' also excludes O2 completely from the binding pocket. Previous studies have shown that the NO binding mechanism is regulated by a proximal arginine residue (R124) and a distal leucine residue (L16). Here, we have investigated the roles of these residues in maintaining the affinity for NO in the heme binding environment by using various time-resolved spectroscopy techniques that span the entire femtosecond-second temporal range in the UV-vis spectrum, and the femtosecond-nanosecond range by IR spectroscopy. Our findings indicate that the tightly regulated NO rebinding events following excitation in wild-type cytochrome c' are affected in the R124A variant. In the R124A variant, vibrational and electronic changes extend continuously across all time scales (from fs-s), in contrast to wild-type cytochrome c' and the L16A variant. Based on these findings, we propose a NO (re)binding mechanism for the R124A variant of cytochrome c' that is distinct from that in wild-type cytochrome c'. In the wider context, these findings emphasize the importance of heme pocket architecture in maintaining the reactivity of hemoproteins towards their chosen ligand, and demonstrate the power of spectroscopic probes spanning a wide temporal range.

Microrheology of Highly Crosslinked Microtubule Networks is Dominated by Force-Induced Crosslinker Unbinding

We determine time- and force-dependent viscoelastic responses of reconstituted networks of microtubules that have been strongly crosslinked by biotin-streptavidin bonds. To measure the microscale viscoelasticity of such networks, we use a magnetic tweezers device to apply localized forces. At short time scales, the crosslinked networks respond nonlinearly to applied force, with stiffening at small forces and softening at high forces, which we attribute to the force-induced unbinding of crosslinks. At long time scales, force-induced bond unbinding leads to local network rearrangement and significant bead creep. Interestingly, the network retains its elastic modulus even under conditions of significant plastic flow, suggesting that crosslinker breakage is balanced by the formation of new bonds. To better understand this effect, we developed a finite element model of such a stiff filament network with labile crosslinkers obeying force dependent Bell model unbinding dynamics. We confirm that for rigid MT filaments having many crosslinks, the coexistence of dissipation and elastic recovery of the network is possible as a result of bond unbinding and rebinding events. Elastic recovery can occur as long as a sufficient number of the original crosslinkers are preserved under the loading period. Plastic flow increases with the decreasing fraction of original crosslinkers preserved. It is interesting to note that the dissipative and plastic responses of such networks to applied loads are more similar to crack propagation in solids than to standard polymer rheology, where standard mechanisms for energy dissipation are hydrodynamics, filament contour fluctuations, etc. Our results will have important implications for understanding mechanical properties of cytoskeletons, where networks of MTs and Factin bundles crosslinked by different flexible and transient crosslinks are locally deformed by transport of intracellular cargos and by the large-scale structural changes in cell division, motility and morphogenesis.

Core-shell nanostructured molecular imprinting fluorescent chemosensor for selective detection ofatrazine herbicide

To convert the binding events on molecularly imprinted polymers (MIPs) into physically detectable signals and to extract the templates completely are the great challenges in developing MIP-based sensors. In this paper, a core-shell nanostructure was employed in constructing the MIP chemosensor for the improvements of template extraction efficiency and imprinted sites accessibility. Vinyl-substituted zinc(II) protoporphyrin (ZnPP) was used as both fluorescent reporter and functional monomer to synthesize atrazine-imprinted polymer shell at silica nanoparticle cores. The template atrazine coordinates with the Lewis acid binding site Zn of ZnPP to form a complex for the molecular imprinting polymerization. These imprinted sites are located in polymer matrix of the thin shells (~8 nm), possessing better accessibility and lower mass-transfer resistance for the target molecules. The fluorescence properties of ZnPP around the imprinted sites will vary upon rebinding of atrazine to these imprinted sites, realizing the conversion of rebinding events into detectable signals by monitoring fluorescence spectra. This MIP probe showed a limit of detection (LOD) of about 1.8 μM for atrazine detection. The core-shell nanostructured MIP method not only improves the sensitivity, but also shows high selectivity for atrazine detection when compared with the non-molecular imprinted counterparts.

Coupled Mesoscopic and Microscopic Simulation of Stochastic Reaction-Diffusion Processes in Mixed Dimensions

We present a new simulation algorithm that allows for dynamic switching between a mesoscopic and a microscopic modeling framework for stochastic reaction-diffusion kinetics. The more expensive and more accurate microscopic model is used only for those species and in those regions in space where there is reason to believe that a microscopic model is needed to capture the dynamics correctly. The microscopic algorithm is extended to simulation on curved surfaces in order to model reaction and diffusion on membranes. The accuracy of the method on and near a spherical membrane is analyzed and evaluated in a numerical experiment. Two biologically motivated examples are simulated in which the need for microscopic simulation of parts of the system arises for different reasons. First, we apply the method to a model of the phosphorylation reactions in a mitogen-activated protein kinase signaling cascade where microscale methods are necessary to resolve fast rebinding events. Then a model is considered for transport of a species over a membrane coupled to reactions in the bulk. The new algorithm attains an accuracy similar to a full microscopic simulation by handling critical interactions on the microscale, but at a significantly reduced cost by using the mesoscale framework for most parts of the biological model.