Crowding Agents Research Articles

Denaturation studies of Clarias gariepinus glutathione transferase in dilute and crowded solutions.

It is important to understand the effect of crowding conditions on the native structure and functional state of enzymes. Equilibrium denaturation studies of Clarius gariepinus GST (CgGST) by guanidine hydrochloride (GdHCl) under dilute conditions and in separate solutions of 0-100gdm-3 Ficoll 70, polyethylene glycol 6000 (PEG 6000) and equal w/v mixtures of the two polymers at 25°C and pH 7.4 were studied fluorometrically. The data were analyzed based on a two-state model assuming the native protein dimer separates into two monomers and then unfolds. The standard free energy of unfolding (ΔG°UN) increases with increasing concentration of each crowding agent in a manner suggesting that high concentrations of PEG 6000 and Ficoll 70 favour the native CgGST relative to the unfolded form. Ficoll 70 stabilizes the native CgGST better than PEG 6000 at low w/v concentration. A mixture of equal g/cm3 concentrations of both crowding agents, however, stabilizes the native form more effectively than either Ficoll 70 or PEG 6000 at equivalent w/v total concentration and is less sensitive to GdHCl. This is in strong agreement with the results of refolding studies, and suggests that a mixture of molecular crowders of widely different molecular weights might show enhanced excluded volume effects compared to a single crowder. Thus, mixed crowding agents more effectively protect the enzyme against denaturation and assist in renaturation better than a single crowder. This suggests a heterogeneous solution of crowders, as will be found within cells, enhances the beneficial effect of crowding on the folded protein stability.

An equation for biomimicking macromolecular crowding using Escherichia coli MG1655 strain

Macromolecules present in the intracellular environment of a cell are densely packed, resulting in a highly crowded cytosolic environment. This crowded milieu influences several biochemical equilibria such as diffusibility and association constant of biomolecules which impose a serious impact on cellular functions as well as its processes. A number of in silico and in vitro studies have been reported till date about using synthetic crowding agents for resembling such a crowding environment within the cell. Lately, it has been realized that synthetic crowders are not suitable for mimicking the intrinsic environment of the cell. In this study, proteins were assumed to be the major biological molecule which contributes to the crowding environment. We have semi-theoretically determined the total protein concentration within an individual E. coli MG1655 cell which changes notably as the growth curve proceeds from 0.2 to 1.0 OD600. The average range of total cellular protein concentration throughout the batch culture was found to be in the range of 15.2 to 178 fg/fL of cytoplasmic volume. The fundamental knowledge gained through the study was translated to applied research in the form of an equation. We propose an equation that could help to mimic the OD600 dependent crowding environment present within a single cell of E. coli in the desired volume of reaction solution. In a nutshell, the equation provides quantitative estimation of the volume of culture required to prepare the cell lysate for biomimicking the intracellular crowding environment in vitro. This finding provides a new insight into the cellular cytosolic environment that could be used as a platform to frame more cells like environment in cell-free protein synthesis (CFPS) system for synthetic biology applications.

Mixture of Macromolecular Crowding Agents Has a Non-additive Effect on the Stability of Proteins.

The folding and unfolding of proteins inside a cell take place in the presence of macromolecules of various shapes and sizes. Such crowded conditions can significantly affect folding, stability, and biophysical properties of proteins. Thus, to logically mimic the intracellular environment, the thermodynamic stability of two different proteins (lysozyme and α-lactalbumin) was investigated in the presence of mixtures of three crowding agents (ficoll 70, dextran 70, and dextran 40) at different pH values. These crowders possess different shapes and sizes. It was observed that the stabilizing effect of mixtures of crowders is more than the sum effects of the individual crowder, i.e., the stabilizing effect is non-additive in nature. Moreover, dextran 40 (in the mixture) has been found to exhibit the greatest stabilization when compared with other crowders in the mixture. In other words, the small size of the crowder has been observed to be a dominant factor in stabilization of the proteins. Graphical Abstract.

Thermodynamic characterization and nearest neighbor parameters for RNA duplexes under molecular crowding conditions.

It is essential to study RNA under molecular crowding conditions to better predict secondary structures of RNAs in vivo. No systematic study has been completed to determine the effects of molecular crowding on RNA duplexes of varying lengths and sequence composition. Here, optical melting, circular dichroism, and osmometry data were collected for RNA duplexes in a 20% polyethylene glycol (with an average molecular weight of 200 g/mol) solution (PEG 200), and nearest neighbor parameters were derived using this data. RNA duplexes are destabilized, on average, 1.02 kcal/mol in the presence of 20% PEG 200. The ΔG°37 values predicted by the nearest neighbor parameters for RNA duplexes in 20% PEG 200 were ∼0.65 kcal/mol closer to experimental ΔG°37 values than those predicted by the standard nearest neighbor model. For one DNA sequence in solution with small crowders, the ΔG°37 values predicted by the 20% PEG 200 RNA nearest neighbor parameters were closer to the experimental values than ΔG°37 values predicted by either the RNA or DNA standard nearest neighbor models. This indicates that the nearest neighbor parameters for RNA duplexes in 20% PEG 200 may be generalizable to RNA and DNA duplexes in solutions with small crowding agents.

Position-based real-time simulation of large crowds

We introduce a crowd simulation method that runs at interactive rates for on the order of a hundred thousand agents, making it particularly suitable for use in games. Our new method is inspired by Position-Based Dynamics (PBD), a fast physics-based animation technique in widespread use. Individual agents in crowds are abstracted by particles, whose motions are controlled by intuitive position constraints and planning velocities, which can be readily integrated into a standard PBD solver, and agent positions are projected onto constraint manifolds to eliminate colliding configurations. A variety of constraints are presented, enabling complex collective behaviors with robust agent collision avoidance in both sparse and dense crowd scenarios.

Effect of Molecular Crowding Agents on the Activity and Stability of Immunosuppressive Enzyme Indoleamine 2,3‐Dioxygenase 1

AbstractIndoleamine 2,3‐dioxygenase 1 (IDO1) plays pivotal role in regulating the metabolism of L‐tryptophan (L‐Trp) through kynurenine pathway, which is a well‐established therapeutic target for the treatment of diseases associated with immunosuppression. Disproportionate expression of the enzyme and poor prediction of various types of malignancies make the development and clinical trials of IDO1 inhibitors extremely relevant. However, IDO1‐based drug development has been hindered due to the use of idealized dilute buffer solution media during the screening and optimization of inhibitors, which do not reflect the highly crowded intracellular environment. In this report, IDO1 enzyme activity and its inhibitory activity against few potent compounds of N′‐hydroxyamidine and aryl‐substituted pyran scaffolds were investigated under macromolecularly crowded environment. The synthetic crowding agents were used to generate a cellular mimetic condition where IDO1 enzyme was found to retain its activity. A non‐linear relationship between the size and volume of the crowding agent with enzyme kinetic parameters was observed. The results suggest that judicious use of size and volume of the crowding agent could provide cellular mimetic environment. A very similar catalytic efficiency, inhibitory activity along with preservation of secondary structural content of IDO1 enzyme under the selected crowded media makes these crowding agents appropriate for further therapeutic application.

Microtubule Self-Organization in the Presence of Crowding Agents

Microtubule Self-Organization in the Presence of Crowding Agents

The Polydispersity Problem: Investigating the Effect of Crowding Agent Polydispersity in Protein Stability

The Polydispersity Problem: Investigating the Effect of Crowding Agent Polydispersity in Protein Stability

Pulsatile Gating of Giant Vesicles Containing Macromolecular Crowding Agents Induced by Colligative Nonideality.

The ability of large macromolecules to exhibit nontrivial deviations in colligative properties of their aqueous solutions is well-appreciated in polymer physics. Here, we show that this colligative nonideality subjects giant lipid vesicles containing inert macromolecular crowding agents to osmotic pressure differentials when bathed in small-molecule osmolytes at comparable concentrations. The ensuing influx of water across the semipermeable membrane induces characteristic swell-burst cycles: here, cyclical and damped oscillations in size, tension, and membrane phase separation occur en route to equilibration. Mediated by synchronized formation of transient pores, these cycles orchestrate pulsewise ejection of macromolecules from the vesicular interior reducing the osmotic differential in a stepwise manner. These experimental findings are fully corroborated by a theoretical model derived by explicitly incorporating the contributions of the solution viscosity, solute diffusivity, and the colligative nonideality of the osmotic pressure in a previously reported continuum description. Simulations based on this model account for the differences in the details of the noncolligatively induced swell-burst cycles, including numbers and periods of the repeating cycles, as well as pore lifetimes. Taken together, our observations recapitulate behaviors of vesicles and red blood cells experiencing sudden osmotic shocks due to large (hundreds of osmolars) differences in the concentrations of small molecule osmolytes and link intravesicular macromolecular crowding with membrane remodeling. They further suggest that any tendency for spontaneous overcrowding in single giant vesicles is opposed by osmotic stresses and requires independent specific interactions, such as associative chemical interactions or those between the crowders and the membrane boundary.

Torso Crowds.

We present a novel dense crowd simulation method. In real crowds of high density, people manoeuvring the crowd need to twist their torso to pass between others. Our proposed method does not use the traditional disc-shaped agent, but instead employs capsule-shaped agents, which enables us to plan such torso orientations. Contrary to other crowd simulation systems, which often focus on the movement of the entire crowd, our method distinguishes between active agents that try to manoeuvre through the crowd, and passive agents that have no incentive to move. We introduce the concept of a focus point to influence crowd agent orientation. Recorded data from real human crowds are used for validation, which shows that our proposed model produces equivalent paths for 85 percent of the validation set. Furthermore, we present a character animation technique that uses the results from our crowd model to generate torso-twisting and side-stepping characters.

Insoluble Off-Pathway Aggregates as Crowding Agents during Amyloid Fibril Formation.

The study of drug candidates for the treatment of amyloidosis and neurodegenerative diseases frequently involves in vitro measurements of amyloid fibril formation. Macromolecular crowding and off-pathway aggregation (OPA) are, by different reasons, two important phenomena affecting the scalability of amyloid inhibitors and their successful application in vivo. On the one hand, the cellular milieu is crowded with macromolecules that drastically increase the effective (thermodynamic) concentration of the amyloidogenic protein. On the other hand, off-pathway aggregates, rather than amyloid fibrils, are increasingly appointed as causative agents of toxicity. The present contribution reveals that insoluble off-pathway aggregates of hen egg-white lysozyme (HEWL) are a peculiar type of crowding agents that, unlike classical macromolecular crowders, decrease the thermodynamic concentration of protein. Illustrating this effect, OPA is shown to resume after lowering the fraction of insoluble aggregates at a constant soluble HEWL concentration. Protein depletion and thioflavin-T fluorescence progress curves indicate that OPA rebirth is not accompanied by additional amyloid fibril formation. The crystallization-like model extended to account for OPA and time-dependent activity coefficients is able to fit multiple kinetic results using a single set of three parameters describing amyloid nucleation, autocatalytic growth, and off-pathway nucleation. The list of fitted results notably includes the cases of aggregation rebirth and all types of progress curves measured for different HEWL concentrations. The quantitative challenges posed by macromolecular crowding and OPA find here a unified response with broader implications for the development of on- and off-pathway inhibitors.

Translational Dynamics of Lipidated Ras Proteins in the Presence of Crowding Agents and Compatible Osmolytes.

Ras proteins are small GTPases and are involved in transmitting signals that control cell growth, differentiation, and proliferation. Since the cell cytoplasm is crowded with different macromolecules, understanding the translational dynamics of Ras proteins in crowded environments is crucial to yielding deeper insight into their reactivity and function. Herein, the translational dynamics of lipidated N-Ras and K-Ras4B is studied in the bulk and in the presence of a macromolecular crowder (Ficoll) and the compatible osmolyte and microcrowder sucrose by fluorescence correlation spectroscopy. The results reveal that N-Ras forms dimers due to the presence of its lipid moiety in the hypervariable region, whereas K-Ras4B remains in its monomeric form in the bulk. Addition of a macromolecular crowding agent gradually favors clustering of the Ras proteins. In 20 wt % Ficoll N-Ras forms trimers and K-Ras4B dimers. Concentrations of sucrose up to 10 wt % foster formation of N-Ras trimers and K-Ras dimers as well. The results can be rationalized in terms of the excluded-volume effect, which enhances the association of the proteins, and, for the higher concentrations, by limited-hydration conditions. The results of this study shed new light on the association state of these proteins in a crowded environment. This is of particular interest for the Ras proteins, because their solution state-monomeric or clustered-influences their membrane-partitioning behavior and their interplay with cytosolic interaction partners.

The Effect of Milk Constituents and Crowding Agents on Amyloid Fibril Formation by κ-Casein.

When not incorporated into the casein micelle, κ-casein, a major milk protein, rapidly forms amyloid fibrils at physiological pH and temperature. In this study, the effects of milk components (calcium, lactose, lipids, and heparan sulfate) and crowding agents on reduced and carboxymethylated (RCM) κ-casein fibril formation was investigated using far-UV circular dichroism spectroscopy, thioflavin T binding assays, and transmission electron microscopy. Longer-chain phosphatidylcholine lipids, which form the lining of milk ducts and milk fat globules, enhanced RCM κ-casein fibril formation irrespective of whether the lipids were in a monomeric or micellar state, whereas shorter-chain phospholipids and triglycerides had little effect. Heparan sulfate, a component of the milk fat globule membrane and catalyst of amyloid deposition in extracellular tissue, had little effect on the kinetics of RCM κ-casein fibril formation. Major nutritional components such as calcium and lactose also had no significant effect. Macromolecular crowding enhances protein-protein interactions, but in contrast to other fibril-forming species, the extent of RCM κ-casein fibril formation was reduced by the presence of a variety of crowding agents. These data are consistent with a mechanism of κ-casein fibril formation in which the rate-determining step is dissociation from the oligomer to give the highly amyloidogenic monomer. We conclude that the interaction of κ-casein with membrane-associated phospholipids along its secretory pathway may contribute to the development of amyloid deposits in mammary tissue. However, the formation of spherical oligomers such as casein micelles is favored over amyloid fibrils in the crowded environment of milk, within which the occurrence of amyloid fibrils is low.

β-Hairpin Crowding Agents Affect α-Helix Stability in Crowded Environments.

The dense, heterogeneous cellular environment is known to affect protein stability. It is now recognized that attractive "quinary" interactions with other biomacromolecules in the cell, referred to as the crowding agents, play a significant role in determining the stability of the protein of interest or test protein. These attractive interactions can reduce or overcome the stabilizing effect of the excluded volume of the crowding agents. However, the roles of specific interactions, such as hydrogen bonding and side chain-side chain hydrophobic interactions, are still unclear. Here, we use molecular simulation to investigate the roles played by hydrophobic interactions and hydrogen bonding between a small helical test protein and equally sized crowding agent proteins in a fixed β-hairpin configuration. The test protein and crowding agents are represented by a coarse-grained protein model, and we use multicanonical molecular dynamics to study the folding thermodynamics of the test protein. Our results confirm that the stability of the test protein depends on the hydrophobicity of the crowding agents and that the stability of the test protein is reduced through favorable side chain-side chain interactions that preferentially stabilize the unfolded states. In addition, we show that when the intermolecular hydrophobic interactions are more favorable than the intramolecular hydrophobic interactions, the β-rich crowding agents can completely destabilize the test protein, causing it to adopt configurations with increased β-content and preventing it from forming its native helical state. Similarities between our results and those seen in the formation of amyloid fibrils are also discussed.

Chemical crowd control agents.

Chemical crowd control agents are also referred to as riot control agents and are mainly used by civil authorities and government agencies to curtail civil disobedience gatherings or processions by large crowds. Common riot control agents used to disperse large numbers of individuals into smaller, less destructive, and more easily controllable numbers include chloroacetophenone, chlorobenzylidenemalononitrile, dibenzoxazepine, diphenylaminearsine, and oleoresin capsicum. In this paper, we discuss the emergency medical care needed by sufferers of acute chemical agent contamination and raise important issues concerning toxicology, safety and health.

Do Macromolecular Crowding Agents Exert Only an Excluded Volume Effect? A Protein Solvation Study.

The effect of macromolecular crowding on protein structure and dynamics has mostly been explained on the basis of the excluded volume effect, its origin being entropic. In recent times a progressive shift in this view has been taking place with increasing emphasis on soft interactions that are enthalpic by nature. Using very low concentrations (1-10 g/L) of both synthetic (dextran- and poly(ethylene glycol) (PEG)-based) and protein (α-synuclein and myoglobin)-based crowders, we have shown that the solvation of probe molecule ANS (1-anilinonapthalene-8-sulfonate) bound to serum proteins bovine serum albumin (BSA) and human serum albumin (HSA) is significantly modulated in both a protein- and crowder-dependent fashion. Since under such conditions the effect of excluded volume is appreciably low, we propose that our observations are direct evidence of soft interactions between the macromolecular crowding agents used and the serum proteins. Moreover, our data reveal, that since at these low crowder concentrations major perturbations to the protein structure are unlikely to take place while minor perturbations might not be readily visible, protein solvation provides a unique spectral signature for capturing such local dynamics, thereby allowing one to decouple hard-sphere interactions from soft sphere ones. Furthermore, since fast fluctuations are known to play a major role in determining the functional characteristics of proteins and enzymes, our results suggest that such motions are prone to be modulated even when the cellular crowding conditions are quite relaxed. In other words, by the time the excluded volume effects come into the picture in the physiological milieu, modulations of functionally important protein motions that need a relatively lower activation energy have already taken place as a result of the aforementioned enthalpic (soft) interactions.

Challenge of mimicking the influences of the cellular environment on RNA structure by PEG-induced macromolecular crowding.

There are large differences between the cellular environment and the conditions widely used to study RNA in vitro. SHAPE RNA structure probing in Escherichia coli cells has shown that the cellular environment stabilizes both long-range and local tertiary interactions in the adenine riboswitch aptamer domain. Synthetic crowding agents are widely used to understand the forces that stabilize RNA structure and in efforts to recapitulate the cellular environment under simplified experimental conditions. Here, we studied the structure and ligand binding ability of the adenine riboswitch in the presence of the macromolecular crowding agent, polyethylene glycol (PEG). Ethylene glycol and low-molecular mass PEGs destabilized RNA structure and caused the riboswitch to sample secondary structures different from those observed in simple buffered solutions or in cells. In the presence of larger PEGs, longer-range loop-loop interactions were more similar to those in cells than in buffer alone, consistent with prior work showing that larger PEGs stabilize compact RNA states. Ligand affinity was weakened by low-molecular mass PEGs but increased with high-molecular mass PEGs, indicating that PEG cosolvents exert complex chemical and steric effects on RNA structure. Regardless of polymer size, however, nucleotide-resolution structural characteristics observed in cells were not recapitulated in PEG solutions. Our results reveal that the cellular environment is difficult to recapitulate in vitro; mimicking the cellular state will likely require a combination of crowding agents and other chemical species.

Separation of Epigallocatechin Gallate from Natural Plant Extracts Using Crowding Agents—Assisted Imprinted Polymers

A methodology-based molecularly imprinted polymer (MIP) was proposed to separate epigallocatechin gallate (EGCG) from natural plant extracts. A molecular crowding agent was introduced to improve the affinity and recognizing ability of EGCG in the preparation of an MIP monolith. Acrylamide was used as the functional monomer, and ethylene glycol dimethacrylate was the crosslinking monomer, using a solution of polystyrene in tetrahydrofuran as a molecular crowding agent with isooctane as a coporogen. In addition, it was found that the ratio of the macromolecule agent, the amount of templates and crosslinking degree, and the composition of the mobile phase greatly affected the retention of the template and performance of molecular recognition. The imprinting factor of the resulting MIP monolith obtained was up to 9.67. The resulting MIP can be used to purify EGCG from crude tea polyphenol efficiently with mean recoveries of 87.42 %.

Synergistic rate boosting of collagen fibrillogenesis in heterogeneous mixtures of crowding agents.

The competition for access to space that arises between macromolecules is the basis of the macromolecular crowding phenomenon, known to modulate biochemical reactions in subtle ways. Crowding is a highly conserved physiological condition in and around cells in metazoans, and originates from a mixture of heterogeneous biomolecules. Here, using collagen fibrillogenesis as an experimental test platform and ideas from the theory of nonideal solutions, we show that an entropy-based synergy is created by a mixture of two different populations of artificial crowders, providing small crowders with extra volume occupancy when in the vicinity of bigger crowders. We present the physiological mechanism by which synergistic effects maximize volume exclusion with the minimum amount of heterogeneous crowders, demonstrating how the evolutionarily optimized crowded conditions found in vivo can be reproduced effectively in vitro.

The effect of Arg on the structure perturbation and chaperone activity of α-crystallin in the presence of the crowding agent, dextran.

α-Crystallin is a protein that is expressed at high levels in all vertebrate eye lenses. It has a molecular weight of 20 kDa and is composed of two subunits: αA and αB. α-Crystallin is a member of the small heat shock protein (sHsps) family that has been shown to prevent protein aggregation. Small molecules are organic compounds that have low molecular weight (<800 Da). Arginin (Arg) is a small molecule and has been shown to prevent protein aggregation through interaction with partially folded intermediates. In this study, the effect of Arg on the chaperone activity of α-crystallin in the presence of dextran, as a crowding agent, against ordered and disordered aggregation of different target proteins (α-lactalbumin, ovotransferrin, and catalase) has been investigated. The experiments were done using visible absorption spectroscopy, ThT-binding assay, fluorescence spectroscopy, and CD spectroscopy. The results showed that in amorphous aggregation and amyloid fibril formation, both in the presence and absence of dextran, Arg had a positive effect on the chaperone action of α-crystallin. However, in the presence of dextran, the effect of Arg on the chaperone ability of α-crystallin was less than in its absence. Thus, our result suggests that crowding interior media decreases the positive effect of Arg on the chaperone ability of α-crystallin. This is a very important issue, since we are trying to find a mechanism to protect living cells against the toxic effect of protein aggregation.