Harnessing Water Competition to Drive Enzyme Crosstalk.

In nature, enzymatic pathways often involve compartmentalization effects that can modify the intrinsic activity and specificity of the different enzymes involved. Consequently, extensive research has focused on replicating and studying the compartmentalization effects on individual enzymes and on multistep enzyme "cascade" reactions. This study explores the influence of compartmentalization achieved using molecular crowding on the glucose oxidase/horseradish peroxidase (GOx/HRP) cascade reaction. The crowder tested is methoxy poly(ethylene glycol) (mPEG) that can, depending on conditions, promote GOx and HRP coassociation at the nanoscale and extend their contact time. Low-molecular-weight mPEG (0.35 kDa), but not mPEG of higher molecular weights (5 or 20 kDa), significantly enhanced the cascade reaction where up to a 20-fold increase in the rate of the cascade reaction was observed under some conditions. The combined analyses emphasize the particularity of low-molecular-weight mPEG and point toward mPEG-induced coassociation of HRP and GOx, producing nearest crowded neighbor effects of HRP on GOx, and vice versa. These altered the nanoscale environments of these enzymes, which influenced substrate affinity. Using mPEG to promote protein coassociation is simple and does not chemically modify the proteins studied. This approach could be of interest for more broadly characterizing nearest crowded neighbor effects (i.e., protein-protein interactions) for multiprotein systems (i.e., more than just two), thus making it an interesting tool for studying very complex systems, such as those found in nature.

Overcoming PEG─Protein Mutual Repulsion to Improve the Efficiency of PEGylation.

Bioconjugation reactions, such as protein PEGylation, generally require excess reagents because of their inefficiency. Intriguingly, few reports have investigated the fundamental causes of this inefficiency. This study demonstrates that the excluded volume effect (EVE)─caused by the mutual repulsion of methoxy poly(ethylene glycol) (mPEG) and proteins under typical PEGylation conditions─causes proteins and protein-reactive mPEG (5 kDa) to self-associate into separate "protein-rich" and "mPEG-rich" nano-domains (i.e., soluble self-assemblies). To overcome this obstacle to reaction, "unreactive" low-molecular-weight mPEG was added as a co-solvent to promote the association between the larger protein and the reactive mPEG molecules by harnessing the same EVE. The near complete PEGylation of lysozyme could be achieved with close to stoichiometric amounts of reactive mPEG, and beneficial effects were observed for other proteins. Considering the general nature of the EVE (e.g., salting-out and PEGying-out), this study provides important perspectives on enhancing bioconjugation reactions, which are relevant to many nanoscale systems.

Electrophoretic ratcheting of spherical particles in well/channel microfluidic devices: Making particles move against the net field.

We examine the electrophoresis of spherical particles in microfluidic devices made of alternating wells and narrow channels, including a system previously used to separate DNA molecules. Our computer simulations predict that such systems can be used to separate spherical particles of different sizes that share the same free-solution mobility. Interestingly, the electrophoretic velocity shows an inversion as the field intensity is increased: while small particles have higher velocities at low field, the situation is reversed at high fields with the larger particles then moving faster. The resulting nonlinearity suggests that asymmetric pulsed electric fields could be used to build separation ratchets: particles then have a net size-dependent velocity in the presence of a zero-mean external field. Exploiting the inversion mentioned above, we show how to design pulsed field sequences that make particles move against the mean field (an example of negative mobility). Finally, we demonstrate that it is possible to use pulsed fields to make particles of different sizes move in opposite directions, even though their charge have the same sign.

Ultra-sub-stoichiometric "Dynamic" Bioconjugation Reduces Viscosity by Disrupting Immunoglobulin Oligomerization.

Monoclonal antibodies (mAb) are a major focus of the pharmaceutical industry, and polyclonal immunoglobulin G (IgG) therapy is used to treat a wide variety of health conditions. As some individuals require mAb/IgG therapy their entire life, there is currently a great desire to formulate antibodies for bolus injection rather than infusion. However, to achieve the required doses, very concentrated antibody solutions may be required. Unfortunately, mAb/IgG self-assembly at high concentration can produce an unacceptably high viscosity for injection. To address this challenge, this study expands the concept of "dynamic covalent chemistry" to "dynamic bioconjugation" in order to reduce viscosity by interfering with antibody-antibody interactions. Ultra-sub-stoichiometric amounts of dynamic PEGylation agents (down to the nanomolar) significantly reduced the viscosity of concentrated antibody solutions by interfering with oligomerization.

DNA Translocations through Nanopores under Nanoscale Preconfinement.

To reduce unwanted variation in the passage speed of DNA through solid-state nanopores, we demonstrate nanoscale preconfinement of translocating molecules using an ultrathin nanoporous silicon nitride membrane separated from a single sensing nanopore by a nanoscale cavity. We present comprehensive experimental and simulation results demonstrating that the presence of an integrated nanofilter within nanoscale distances of the sensing pore eliminates the dependence of molecular passage time distributions on pore size, revealing a global minimum in the coefficient of variation of the passage time. These results provide experimental verification that the inter- and intramolecular passage time variation depends on the conformational entropy of each molecule prior to translocation. Furthermore, we show that the observed consistently narrower passage time distributions enables a more reliable DNA length separation independent of pore size and stability. We also demonstrate that the composite nanofilter/nanopore devices can be configured to suppress the frequency of folded translocations, ensuring single-file passage of captured DNA molecules. By greatly increasing the rate at which usable data can be collected, these unique attributes will offer significant practical advantages to many solid-state nanopore-based sensing schemes, including sequencing, genomic mapping, and barcoded target detection.

Molecular Characterization of Binding Loop E in the Nematode Cys-Loop GABA Receptor.

Nematodes exhibit a vast array of cys-loop ligand-gated ion channels with unique pharmacologic characteristics. However, many of the structural components that govern the binding of various ligands are unknown. The nematode cys-loop GABA receptor uncoordinated 49 (UNC-49) is an important receptor found at neuromuscular junctions that plays an important role in the sinusoidal movement of worms. The unique pharmacologic features of this receptor suggest that there are structural differences in the agonist binding site when compared with mammalian receptors. In this study, we examined each amino acid in one of the main agonist binding loops (loop E) via the substituted cysteine accessibility method (SCAM) and analyzed the interaction of various residues by molecular dynamic simulations. We found that of the 18 loop E mutants analyzed, H142C, R147C, and S157C had significant changes in GABA EC50 and were accessible to modification by a methanethiosulfonate reagent (MTSET) resulting in a change in I GABA In addition, the residue H142, which is unique to nematode UNC-49 GABA receptors, appears to play a negative role in GABA sensitivity as its mutation to cysteine increased sensitivity to GABA and caused the UNC-49 receptor partial agonist 5-aminovaleric acid (DAVA) to behave as a full agonist. Overall, this study has revealed potential differences in the agonist binding pocket between nematode UNC-49 and mammalian GABA receptors that could be exploited in the design of novel anthelmintics.

Buckling Causes Nonlinear Dynamics of Filamentous Viruses Driven through Nanopores.

Measurements and Langevin dynamics simulations of filamentous viruses driven through solid-state nanopores reveal a superlinear rise in the translocation velocity with driving force. The mobility also scales with the length of the virus in a nontrivial way that depends on the force. These dynamics are consequences of the buckling of the leading portion of a virus as it emerges from the nanopore and is put under compressive stress by the viscous forces it encounters. The leading tip of a buckled virus stalls and this reduces the total viscous drag force. We present a scaling theory that connects the solid mechanics to the nonlinear dynamics of polyelectrolytes translocating nanopores.

Morphology of depletant-induced erythrocyte aggregates.

Red blood cells suspended in quiescent plasma tend to aggregate into multicellular assemblages, including linearly stacked columnar rouleaux, which can reversibly form more complex clusters or branching networks. While these aggregates play an essential role in establishing hemorheological and pathological properties, the biophysics behind their self-assembly into dynamic mesoscopic structures remains under-explored. We employ coarse-grained molecular simulations to model low-hematocrit erythrocytes subject to short-range implicit depletion forces, and demonstrate not only that depletion interactions are sufficient to account for a sudden dispersion-aggregate transition, but also that the volume fraction of depletant macromolecules controls small aggregate morphology. We observe a sudden transition from a dispersion to a linear column rouleau, followed by a slow emergence of disorderly amorphous clusters of many short rouleaux at larger volume fractions. This work demonstrates how discocyte topology and short-range, non-specific, physical interactions are sufficient to self-assemble erythrocytes into various aggregate structures, with markedly different morphologies and biomedical consequences.

A sequential nanopore-channel device for polymer separation.

In this work, we investigated whether a series of nanopores connected by channels can be used to separate polymer mixtures by molecular size. We conducted multiscale coarse-grained simulations of semiflexible polymers driven through such a device. Polymers were modelled as chains of beads near the nanopores and as single particles in the bulk of the channels. Since polymers rarely escape back into the bulk of the channels after coming sufficiently close to the nanopores, the more computationally expensive simulations near the pores were decoupled from those in the bulk. The distribution of polymer positions after many translocations was deduced mathematically from simulations across a single nanopore-channel pair, under the reasonable assumption of identical and independent dynamics in each channel and each nanopore. Our results reveal rich polymer dynamics in the nanopore-channel device and suggest that it can indeed produce polymer separation. As expected, the mean time to translocate across a single nanopore increases with the chain length. Conversely, the mean time to cross the channels from one nanopore to the next decreases with the chain length, as smaller chains explore more of the channel volume between translocations. As such, the time between translocations is a function of the length and width of the channels. Depending on the channel dimensions, polymers are sorted by increasing length, decreasing length, or non-monotonically by length such that polymers of an intermediate size emerge first.

Sorting polymers by size via an array of viscous posts.

DNA fragments can be sorted according to size by forcing them through an array of nanoposts. Whereas previous studies have explored solid nanoposts, this work examines nanoposts constructed out of viscous inclusions. Langevin dynamics simulations are used to study the dynamics of polymers driven through arrays of these viscous nanoposts for a range of post viscosities. The results are compared to the solid post case. Increasing post viscosity causes a decrease in the mobility of polymers traversing the array. In the limit of high post viscosity, the mobility becomes lower than in the solid post arrays, rather than converging to it. Analysis of the distributions of event times also shows that the viscous case is fundamentally different from the solid post case. The decrease in mobility in the viscous case arises from slowing down the polymer as it interacts with or even moves through the nanoposts, whereas the solid post case exhibits wrapping and unwrapping dynamics, yielding escape-like statistics. This work suggests that it may be possible to use viscous inclusions within nanofluidic and microfluidic devices to sort biomolecules with high resolution.

Modeling and Simulating the Dynamics of Type IV Pili Extension of Pseudomonas aeruginosa.

Bacteria such as Pseudomonas aeruginosa use type IV pili to move across surfaces. The pili extend, attach to the surface, and then retract to move the bacteria forward. In this article, a coarse-grained model of pilus extension and attachment is developed. Simulations performed at biologically relevant conditions indicate that pilus extension is a quasistatic process such that the pili are able to relax via thermal fluctuations as it is being built and extended. Results are generated for pili with different rigidities ranging from very flexible to very stiff. It is shown that very flexible pili do not extend very far and thus would limit the bacteria to short jumps forward while stiff pili enable much greater displacements. Feasible mechanisms of attachment to the surface are also found to vary greatly between flexible and stiff pili. While it is not always the tip of flexible pili that first makes contact with the substrate, it is likely to be a part of the pili that is close to the tip. Conversely, stiff pili are much more likely to make contact with the substrate via the tip, but if not then the part of the pilus that attaches can be quite far from the tip. These results thus give insight to help resolve current discrepancies in the literature regarding pilus stiffness and the location of adhesins on pili.

Translocation Time through a Nanopore with an Internal Cavity Is Minimal for Polymers of Intermediate Length.

The translocation of polymers through nanopores with large internal cavities bounded by two narrow pores is studied via Langevin dynamics simulations. The total translocation time is found to be a nonmonotonic function of polymer length, reaching a minimum at intermediate length, with both shorter and longer polymers taking longer to translocate. The location of the minimum is shown to shift with the magnitude of the applied force, indicating that the pore can be dynamically tuned to favor different polymer lengths. A simple model balancing the effects of entropic trapping within the cavity against the driving force is shown to agree well with simulations. Beyond the nonmonotonicity, detailed analysis of translocation uncovers rich dynamics in which factors such as going to a high force regime and the emergence of a tail for long polymers dramatically change the behavior of the system. These results suggest that nanopores with internal cavities can be used for applications such as selective extraction of polymers by length and filtering of polymer solutions, extending the uses of nanopores within emerging nanofluidic technologies.

Translocation is a nonequilibrium process at all stages: Simulating the capture and translocation of a polymer by a nanopore.

Langevin dynamics simulations of the capture of polymers by a nanopore and the subsequent translocation through the nanopore are performed. These simulations are conducted for several polymer lengths at two different values for the Péclet number, which quantifies the drift-diffusion balance of the system. The capture-translocation process is divided into several stages, and the dynamics of translocation are characterized by measuring the average time for each stage and also the average conformation of the polymer at each stage. Comparison to the standard simulation approach of simulating only the translocation process reveals several important differences. While in the standard protocol, the polymer is essentially equilibrated at the start of translocation, simulations of the capture process reveal a polymer that is elongated when it approaches the pore and either remains elongated or becomes compressed at the start of translocation depending on the drift-diffusion balance. These results demonstrate that translocation is a non-equilibrium process at all stages and that simulations assuming equilibration could yield improper results, even at a qualitative level. The scaling of the translocation time with polymer length is found to be significantly different between the two simulation protocols thus demonstrating that the capture step is an essential part of modeling the translocation process.

Simulating the entropic collapse of coarse-grained chromosomes.

Depletion forces play a role in the compaction and decompaction of chromosomal material in simple cells, but it has remained debatable whether they are sufficient to account for chromosomal collapse. We present coarse-grained molecular dynamics simulations, which reveal that depletion-induced attraction is sufficient to cause the collapse of a flexible chain of large structural monomers immersed in a bath of smaller depletants. These simulations use an explicit coarse-grained computational model that treats both the supercoiled DNA structural monomers and the smaller protein crowding agents as combinatorial, truncated Lennard-Jones spheres. By presenting a simple theoretical model, we quantitatively cast the action of depletants on supercoiled bacterial DNA as an effective solvent quality. The rapid collapse of the simulated flexible chromosome at the predicted volume fraction of depletants is a continuous phase transition. Additional physical effects to such simple chromosome models, such as enthalpic interactions between structural monomers or chain rigidity, are required if the collapse is to be a first-order phase transition.

Translocation of a polymer through a nanopore starting from a confining nanotube.

In this manuscript, Langevin Dynamics simulations and Tension-Propagation theory are used to investigate the forced translocation of a polymer from a confining tube through a nanopore situated at one of the tube's ends. The diameter of the tube allows for a control over the polymer conformations: decreasing the tube diameter reduces the number of conformations available to the polymer chain both before and during translocation. As the tube diameter is decreased, the translocation time is observed to increase. Interestingly, while the width of the distribution of translocation times is reduced if the chain starts in a tube, it reaches a maximum for weakly confining tubes. A Tension-Propagation approach is developed for the tube-nanopore setup in the strongly driven limit. Good agreement between the simulations and the theory allows for an exploration of the underlying physical mechanisms, including the calculation of an effective pore friction and the assessing of the impact of monomer crowding on the trans side.

Coarse-grained molecular dynamics simulations of depletion-induced interactions for soft matter systems.

Given the ubiquity of depletion effects in biological and other soft matter systems, it is desirable to have coarse-grained Molecular Dynamics (MD) simulation approaches appropriate for the study of complex systems. This paper examines the use of two common truncated Lennard-Jones (Weeks-Chandler-Andersen (WCA)) potentials to describe a pair of colloidal particles in a thermal bath of depletants. The shifted-WCA model is the steeper of the two repulsive potentials considered, while the combinatorial-WCA model is the softer. It is found that the depletion-induced well depth for the combinatorial-WCA model is significantly deeper than the shifted-WCA model because the resulting overlap of the colloids yields extra accessible volume for depletants. For both shifted- and combinatorial-WCA simulations, the second virial coefficients and pair potentials between colloids are demonstrated to be well approximated by the Morphometric Thermodynamics (MT) model. This agreement suggests that the presence of depletants can be accurately modelled in MD simulations by implicitly including them through simple, analytical MT forms for depletion-induced interactions. Although both WCA potentials are found to be effective generic coarse-grained simulation approaches for studying depletion effects in complicated soft matter systems, combinatorial-WCA is the more efficient approach as depletion effects are enhanced at lower depletant densities. The findings indicate that for soft matter systems that are better modelled by potentials with some compressibility, predictions from hard-sphere systems could greatly underestimate the magnitude of depletion effects at a given depletant density.

Translocation of "rod-coil" polymers: probing the structure of single molecules within nanopores.

Using simulation and analytical techniques, we demonstrate that it is possible to extract structural information about biological molecules by monitoring the dynamics as they translocate through nanopores. From Langevin dynamics simulations of polymers exhibiting discrete changes in flexibility (rod-coil polymers), distinct plateaus are observed in the progression towards complete translocation. Characterizing these dynamics via an incremental mean first passage approach, the large steps are shown to correspond to local barriers preventing the passage of the coils while the rods translocate relatively easily. Analytical replication of the results provides insight into the corrugated nature of the free energy landscape as well as the dependence of the effective barrier heights on the length of the coil sections. Narrowing the width of the pore or decreasing the charge on either the rod or the coil segments are both shown to enhance the resolution of structural details. The special case of a single rod confined within a nanopore is also studied. Here, sufficiently long flexible sections attached to either end are demonstrated to act as entropic anchors which can effectively trap the rod within the pore for an extended period of time. Both sets of results suggest new experimental approaches for the control and study of biological molecules within nanopores.

Translocation of a polymer through a nanopore modulated by a sticky site.

Using a one-dimensional model for the translocation of a polymer through a nanopore, the effect of a "sticky site" at which the polymer binds to the pore is explored via exact numerical techniques. Results for the mean translocation time and the probability of translocation on the insertion of the first monomer in the pore are generated across a wide range of driving forces and binding potential strengths (well depths). The balance between the driving force, diffusion, and well depth yields a rich set of dynamics that depend strongly on where the sticky site is located along the polymer. For example, when the sticky site is located near the head of the polymer, the translocation time is found to be a maximum at an intermediate driving force with events at lower driving forces taking less time. Additionally, the critical well depth at which the sticky site dominates the dynamics, is found to be a non-monotonic function of the driving force when the sticky site is located at the head or tail of the polymer, but not in the middle. Modeling of the process yields good agreement with simulation results.

Deconvolution volumetric additive manufacturing.

Volumetric additive manufacturing techniques are a promising pathway to ultra-rapid light-based 3D fabrication. Their widespread adoption, however, demands significant improvement in print fidelity. Currently, volumetric additive manufacturing prints suffer from systematic undercuring of fine features, making it impossible to print objects containing a wide range of feature sizes, precluding effective adoption in many applications. Here, we uncover the reason for this limitation: light dose spread in the resin due to chemical diffusion and optical blurring, which becomes significant for features ⪅0.5 mm. We develop a model that quantitatively predicts the variation of print time with feature size and demonstrate a deconvolution method to correct for this error. This enables prints previously beyond the capabilities of volumetric additive manufacturing, such as a complex gyroid structure with variable thickness and a fine-toothed gear. These results position volumetric additive manufacturing as a mature 3D printing method, all but eliminating the gap to industry-standard print fidelity.

Memory effects during the unbiased translocation of a polymer through a nanopore.

Through a detailed Langevin dynamics simulation study, the role of memory effects during unbiased translocation is explored. Tests are devised to uncover the presence of memory effects by directly measuring forward/backward-correlated motion as well as the associated change in the dynamics. Conducting these tests at low and high viscosities, a range of behaviours across different time scales is revealed: short-time forward correlations at all viscosities, quasi-static behaviour at low viscosity, and long-time backward correlations at high viscosity. By applying these tests at different portions of the translocation process, these memory effects are also shown to vary as translocation proceeds. Combining this information with standard measurements, a physical picture of unbiased translocation as the diffusion of a local minimum is proposed.