Jagannath Mondal

When Does Trimethylamine N-Oxide Fold a Polymer Chain and Urea Unfold It?

Longstanding mechanistic questions about the role of protecting osmolyte trimethylamine N-oxide (TMAO) that favors protein folding and the denaturing osmolyte urea are addressed by studying their effects on the folding of uncharged polymer chains. Using atomistic molecular dynamics simulations, we show that 1 M TMAO and 7 M urea solutions act dramatically differently on these model polymer chains. Their behaviors are sensitive to the strength of the attractive dispersion interactions of the chain with its environment: when these dispersion interactions are sufficiently strong, TMAO suppresses the formation of extended conformations of the hydrophobic polymer as compared to water while urea promotes the formation of extended conformations. Similar trends are observed experimentally for real protein systems. Quite surprisingly, we find that both protecting and denaturing osmolytes strongly interact with the polymer, seemingly in contrast with existing explanations of the osmolyte effect on proteins. We show that what really matters for a protective osmolyte is its effective depletion as the polymer conformation changes, which leads to a negative change in the preferential binding coefficient. For TMAO, there is a much more favorable free energy of insertion of a single osmolyte near collapsed conformations of the polymer than near extended conformations. By contrast, urea is preferentially stabilized next to the extended conformation and thus has a denaturing effect.

Entropy-based mechanism of ribosome-nucleoid segregation in E. coli cells.

In Escherichia coli, ribosomes concentrate near the cylindrical wall and at the endcaps, whereas the chromosomal DNA segregates in the more centrally located nucleoid. A simple statistical model recovers the observed ribosome-nucleoid segregation remarkably well. Plectonemic DNA is represented as a hyperbranched hard-sphere polymer, and multiple ribosomes that simultaneously translate the same mRNA strand (polysomes) are represented as freely jointed chains of hard spheres. There are no attractive interactions between particles, only excluded-volume effects. At realistic DNA and ribosome concentrations, segregation arises primarily from two effects: the DNA polymer avoids walls to maximize conformational entropy, and the polysomes occupy the empty space near the walls to maximize translational entropy. In this complex system, maximizing total entropy results in spatial organization of the components. Due to coupling of mRNA to DNA through RNA polymerase, the same entropic effects should favor the placement of highly expressed genes at the interface between the nucleoid and the ribosome-rich periphery. Such a placement would enable efficient cotranscriptional translation and facile transertion of membrane proteins into the cytoplasmic membrane. Finally, in the model, monofunctional DNA polymer beads representing the tips of plectonemes preferentially locate near the cylindrical wall. This suggests that initiation of transcription may occur preferentially near the ribosome-rich periphery.

How osmolytes influence hydrophobic polymer conformations: A unified view from experiment and theory

Significance Osmolytes influence protein structure by either promoting (protecting osmolytes) or disrupting (denaturing osmolytes) the folding process. Current consensus is that protecting osmolytes [trimethylamine N-oxide (TMAO)] act by being excluded from the protein surface while denaturing osmolytes (urea) bind to it. However there is little knowledge about the molecular mechanism of osmolyte action on hydrophobic macromolecules, which form the core of most proteins. This work, through a combination of single-molecule atomic force microscopy experiments and computer simulations, investigates the collapse behavior of a hydrophobic polymer polystyrene in TMAO and urea. The mechanism of osmolyte action on hydrophobic macromolecules is distinct from that of a protein, but, despite key differences, both mechanisms comply with the standard thermodynamic theory of preferential osmolyte binding. It is currently the consensus belief that protective osmolytes such as trimethylamine N-oxide (TMAO) favor protein folding by being excluded from the vicinity of a protein, whereas denaturing osmolytes such as urea lead to protein unfolding by strongly binding to the surface. Despite there being consensus on how TMAO and urea affect proteins as a whole, very little is known as to their effects on the individual mechanisms responsible for protein structure formation, especially hydrophobic association. In the present study, we use single-molecule atomic force microscopy and molecular dynamics simulations to investigate the effects of TMAO and urea on the unfolding of the hydrophobic homopolymer polystyrene. Incorporated with interfacial energy measurements, our results show that TMAO and urea act on polystyrene as a protectant and a denaturant, respectively, while complying with Tanford–Wyman preferential binding theory. We provide a molecular explanation suggesting that TMAO molecules have a greater thermodynamic binding affinity with the collapsed conformation of polystyrene than with the extended conformation, while the reverse is true for urea molecules. Results presented here from both experiment and simulation are in line with earlier predictions on a model Lennard–Jones polymer while also demonstrating the distinction in the mechanism of osmolyte action between protein and hydrophobic polymer. This marks, to our knowledge, the first experimental observation of TMAO-induced hydrophobic collapse in a ternary aqueous system.

Role of water and steric constraints in the kinetics of cavity–ligand unbinding

Significance The unbinding of ligand–substrate systems in molecular water is a problem of great theoretical and practical interest. To understand the dynamical nature of the unbinding, it is desirable to use atomistic techniques like molecular dynamics (MD). However, the associated timescales are typically too long for MD to be applicable. In this work, we apply a recent metadynamics scheme that allows the use of MD to get information on thermodynamics and kinetics of systems that are virtually impossible to treat with unbiased MD. We calculate unbinding pathways and timescales, and show that solvent molecules play a crucial role in cooperation with steric effects. The approaches used here demonstrate a broadly applicable methodology for studying ligand unbinding. A key factor influencing a drug’s efficacy is its residence time in the binding pocket of the host protein. Using atomistic computer simulation to predict this residence time and the associated dissociation process is a desirable but extremely difficult task due to the long timescales involved. This gets further complicated by the presence of biophysical factors such as steric and solvation effects. In this work, we perform molecular dynamics (MD) simulations of the unbinding of a popular prototypical hydrophobic cavity–ligand system using a metadynamics-based approach that allows direct assessment of kinetic pathways and parameters. When constrained to move in an axial manner, the unbinding time is found to be on the order of 4,000 s. In accordance with previous studies, we find that the cavity must pass through a region of sharp wetting transition manifested by sudden and high fluctuations in solvent density. When we remove the steric constraints on ligand, the unbinding happens predominantly by an alternate pathway, where the unbinding becomes 20 times faster, and the sharp wetting transition instead becomes continuous. We validate the unbinding timescales from metadynamics through a Poisson analysis, and by comparison through detailed balance to binding timescale estimates from unbiased MD. This work demonstrates that enhanced sampling can be used to perform explicit solvent MD studies at timescales previously unattainable, to our knowledge, obtaining direct and reliable pictures of the underlying physiochemical factors including free energies and rate constants.

Time‐dependent effects of transcription‐ and translation‐halting drugs on the spatial distributions of the Escherichia coli chromosome and ribosomes

Previously observed effects of rifampicin and chloramphenicol indicate that transcription and translation activity strongly affect the coarse spatial organization of the bacterial cytoplasm. Single‐cell, time‐resolved, quantitative imaging of chromosome and ribosome spatial distributions and ribosome diffusion in live Escherichia coli provides insight into the underlying mechanisms. Monte Carlo simulations of model DNA‐ribosome mixtures support a novel nucleoid‐ribosome mixing hypothesis. In normal conditions, 70S‐polysomes and the chromosomal DNA segregate, while 30S and 50S ribosomal subunits are able to penetrate the nucleoids. Growth conditions and drug treatments determine the partitioning of ribosomes into 70S‐polysomes versus free 30S and 50S subunits. Entropic and excluded volume effects then dictate the resulting chromosome and ribosome spatial distributions. Direct observation of radial contraction of the nucleoids 0–5 min after treatment with either transcription‐ or translation‐halting drugs supports the hypothesis that simultaneous transcription, translation, and insertion of proteins into the membrane (‘transertion’) exerts an expanding force on the chromosomal DNA. Breaking of the DNA‐RNA polymerase‐mRNA‐ribosome‐membrane chain in either of two ways causes similar nucleoid contraction on a similar timescale. We suggest that chromosomal expansion due to transertion enables co‐transcriptional translation throughout the nucleoids.

How hydrophobic drying forces impact the kinetics of molecular recognition

A model of protein–ligand binding kinetics, in which slow solvent dynamics results from hydrophobic drying transitions, is investigated. Molecular dynamics simulations show that solvent in the receptor pocket can fluctuate between wet and dry states with lifetimes in each state that are long enough for the extraction of a separable potential of mean force and wet-to-dry transitions. We present a diffusive surface hopping model that is represented by a 2D Markovian master equation. One dimension is the standard reaction coordinate, the ligand–pocket separation, and the other is the solvent state in the region between ligand and binding pocket which specifies whether it is wet or dry. In our model, the ligand diffuses on a dynamic free-energy surface which undergoes kinetic transitions between the wet and dry states. The model yields good agreement with results from explicit solvent molecular dynamics simulation and an improved description of the kinetics of hydrophobic assembly. Furthermore, it is consistent with a “non-Markovian Brownian theory” for the ligand–pocket separation coordinate alone.

Self-assembly of gemini surfactants: A computer simulation study

The self-assembly behavior of gemini (dimeric or twin-tail) dicarboxylate disodium surfactants is studied using molecular dynamics simulations. A united atom model is employed for the surfactants with fully atomistic counterions and water. This gemini architecture, in which two single tailed surfactants are joined through a flexible hydrophobic linker, has been shown to exhibit concentration-dependent aqueous self-assembly into lyotropic phases including hexagonal, gyroid, and lamellar morphologies. Our simulations reproduce the experimentally observed phases at similar amphiphile concentrations in water, including the unusual ability of these surfactants to form gyroid phases over unprecedentedly large amphiphile concentration windows. We demonstrate quantitative agreement between the predicted and experimentally observed domain spacings of these nanostructured materials. Through careful conformation analyses of the surfactant molecules, we show that the gyroid phase is electrostatically stabilized related to the lamellar phase. By starting with a lamellar phase, we show that use of a bulkier N(CH(3))(4)(+) counterion in place of Na(+) drives the formation of a gyroid phase. Decreasing the charge on the surfactant headgroups by carboxylate protonation decreases the degree of order in the lamellar phase. Using our models, we show that the translational diffusion of water and the Na(+) counterions is decreased by several orders of magnitude over the studied concentration range, and we attribute these effects to strong correlations between the mobile species and the surfactant headgroups.

Sequence-Dependent Interaction of β-Peptides with Membranes

Recent experimental studies have revealed interesting sequence dependence in the antimicrobial activity of β-peptides, which suggests the possibility of a rational design of new antimicrobial agents. To obtain insight into the mechanism of membrane activity, we present a computer simulation study of the adsorption of these molecules to a single-component lipid membrane. Two classes of molecules are investigated: 10-residue oligomers of 14-helical sequences, and four sequences of random copolymeric β-peptides. The oligomers of interest are globally amphiphilic (GA) and nonglobally amphiphilic (non-GA) sequences of 10-residue, 14-helical sequences. In solution and at the interface, all oligomers maintain a helical structure throughout the simulation. The penetration of the molecules into the membrane and the orientation of the molecules at the interface depend strongly on the sequence. We attribute this to the propensity of the β-phenylalanine (βF) residues for membrane penetration. For the four sequences of random copolymeric β-peptides, simulations of an implicit solvent and membrane model show that the strength of adsorption of the polymers is strongly correlated with their efficiency to segregate the hydrophobic and cationic residues. The simulations suggest simple strategies for the design of candidates for antimicrobial β-peptides. Collectively, these results further support the conclusion from several recent studies that neither global amphiphilicity nor regular secondary structure is required for short peptides to effectively interact with the membrane. Moreover, although we study only the binding process, the fact that there is a correlation between the sequence dependence in the calculated binding properties and the experimentally observed antimicrobial activity suggests that efficient binding to the membrane might be a good predictor for high antimicrobial activity.

Mechanically-driven phase separation in a growing bacterial colony

Significance Bacteria self-organize into a dense multicellular community known as a biofilm, in which cells are embedded in self-secreted extracellular polymeric substances (EPSs). A number of processes can contribute to spatial heterogeneity in a growing biofilm; among them, the effect of macromolecular crowding enhanced by the EPSs has largely remained unexplored. To understand the effect of macromolecular crowding in spontaneous spatial organization, we develop a computational model to investigate the explicit role of mechanical interactions in driving the collective behavior of bacterial cells in the presence of EPS particles in a colony growing on a solid substrate. Our findings demonstrate that an entropy-driven depletion interaction between bacteria and EPSs can induce significant phase separation and spatial heterogeneity in a biofilm. Secretion of extracellular polymeric substances (EPSs) by growing bacteria is an integral part of forming biofilm-like structures. In such dense systems, mechanical interactions among the structural components can be expected to significantly contribute to morphological properties. Here, we use a particle-based modeling approach to study the self-organization of nonmotile rod-shaped bacterial cells growing on a solid substrate in the presence of self-produced EPSs. In our simulation, all of the components interact mechanically via repulsive forces, occurring as the bacterial cells grow and divide (via consuming diffusing nutrient) and produce EPSs. Based on our simulation, we show that mechanical interactions control the collective behavior of the system. In particular, we find that the presence of nonadsorbing EPSs can lead to spontaneous aggregation of bacterial cells by a depletion attraction and thereby generates phase separated patterns in the nonequilibrium growing colony. Both repulsive interactions between cell and EPSs and the overall concentration of EPSs are important factors in the self-organization in a nonequilibrium growing colony. Furthermore, we investigate the interplay of mechanics with the nutrient diffusion and consumption by bacterial cells and observe that suppression of branch formation occurs due to EPSs compared with the case where no EPS is produced.

Role of Desolvation in Thermodynamics and Kinetics of Ligand Binding to a Kinase

Computer simulations are used to determine the free energy landscape for the binding of the anticancer drug Dasatinib to its src kinase receptor and show that before settling into a free energy basin the ligand must surmount a free energy barrier. An analysis based on using both the ligand-pocket separation and the pocket-water occupancy as reaction coordinates shows that the free energy barrier is a result of the free energy cost for almost complete desolvation of the binding pocket. The simulations further show that the barrier is not a result of the reorganization free energy of the binding pocket. Although a continuum solvent model gives the location of free energy minima, it is not able to reproduce the intermediate free energy barrier. Finally, it is shown that a kinetic model for the on rate constant in which the ligand diffuses up to a doorway state and then surmounts the desolvation free energy barrier is consistent with published microsecond time-scale simulations of the ligand binding kinetics for this system [Shaw, D. E. et al. J. Am. Chem. Soc.2011, 133, 9181−918321545110].