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Molecularly crowded solutions have been used to simulate cytoplasmic fluids, but little justification is provided in the literature. This work compared three key physical properties of solutions containing polyethylene glycol and sucrose against those in the cytoplasmic fluid from bacteria Escherichia coli: fluid viscosity, relaxation time (T1 and T2) of water, and the diffusion coefficient of water. It was concluded that appropriate mixtures of sucrose and polyethylene glycol 10,000, such as 10% sucrose and 15% PEG10,000 and 7.5% sucrose and 19% PEG10,000, closely mimic these properties of cytoplasmic fluid from E. coli containing around 100 mg/mL of proteins. [ABSTRACT FROM AUTHOR]

The pathological process of prion diseases implicates that the normal physiological cellular prion protein (PrPC) converts into misfolded abnormal scrapie prion (PrPSc) through post-translational modifications that increase β-sheet conformation. We recently demonstrated that HuPrP(90–231) thermal unfolding is partially irreversible and characterized by an intermediate state (β-PrPI), which has been revealed to be involved in the initial stages of PrPC fibrillation, with a seeding activity comparable to that of human infectious prions. In this study, we report the thermal unfolding characterization, in cell-mimicking conditions, of the truncated (HuPrP(90–231)) and full-length (HuPrP(23–231)) human prion protein by means of CD and NMR spectroscopy, revealing that HuPrP(90–231) thermal unfolding is characterized by two successive transitions, as in buffer solution. The amyloidogenic propensity of HuPrP(90–231) under crowded conditions has also been investigated. Our findings show that although the prion intermediate, structurally very similar to β-PrPI, forms at a lower temperature compared to when it is dissolved in buffer solution, in cell-mimicking conditions, the formation of prion fibrils requires a longer incubation time, outlining how molecular crowding influences both the equilibrium states of PrP and its kinetic pathways of folding and aggregation. [ABSTRACT FROM AUTHOR]

A conversion‐chemistry‐based zinc–selenium aqueous battery is reported that delivers high specific capacity, good rate capability, and excellent cycle life. In this work, an electronically conjugated covalent triazine framework is used to physicochemically lock selenium (Se8) clusters. As a control sample, the traditional melt‐diffusion approach is used to physically lock Se8. While the melt‐diffused selenium cathode exhibited a precipitous drop in capacity with cycling, the physicochemically locked selenium cathode can be cycled in a stable manner and delivered a specific capacity of ≈600 mAh g−1 with a capacity retention of ≈70% after 1000 continuous charge/discharge steps. Ab initio density functional theory calculations and various structural and morphological characterizations indicate that the superiority of the physicochemically locked selenium cathode stems from its ability to suppress the polyselenide shuttle phenomenon and thus prevent loss of active material during cycling. This work opens the door toward the development of conversion chemistries for high performing, non‐flammable, and low‐cost zinc‐based rechargeable batteries. [ABSTRACT FROM AUTHOR]

Nanozymes are widely used in various applications as nanosized catalysts for replacing enzymes. An accurate estimation of the catalytic activity of nanozymes in real conditions is critical. In this article, for the first time, we systematically studied the effect of macromolecular molecules co-existing in the real system on the oxidoreductase (peroxidase, oxidase, and catalase)-mimicking nanozymes made of a gold nanoparticle core and a platinum shell, Prussian Blue, Mn2O3 and CoO nanoparticles. Comparisons were made with horseradish peroxidase. We distinguished two main mechanisms of the negative impact of macromolecules on nanozyme catalysis–slowed diffusion and surface shielding of nanoparticles. While the first mechanism is typical for enzymes, the second one is specific only for nanozymes. Understanding the mechanisms is essential for developing approaches to reduce the unavoidable impact of macromolecules for various analytical and biomedical applications. [ABSTRACT FROM AUTHOR]

Aqueous sodium‐ion batteries (ASIBs) have recently emerged as a compelling choice for large‐scale energy storage when considering their safe operational characteristics and cost‐effectiveness. Nonetheless, the inadequate electrochemical stability window (ESW) of water (1.23 V) and severe dissolution of electrodes in aqueous electrolytes have proven bottlenecks to enabling high energy density and long lifespan of ASIBs. Here, a molecular crowding electrolyte is introduced, featuring nonflammable polyethylene glycol dimethyl ether (PED) as a crowding agent and a hydrogen bond receptor to further diminish the water activity. The 4.5 m (mol kg−1) NaClO4‐4 wt% H2O‐96 wt% PED electrolyte displayed an impressive ESW of 3.4 V, all while maintaining a moderate salt concentration of 4.5 m. Meanwhile, this electrolyte demonstrated substantial mitigation of vanadium dissolution in Na3V2(PO4)3. Consequently, in a comprehensive evaluation, the aqueous NaTi2(PO4)3||Na3V2(PO4)3 full cell exhibited an energy density of 47 Wh kg−1 at 1 C with a 74% retention after 100 cycles, and displayed negligible capacity decay (≈0.014% per cycle) at 5 C in 1000 cycles with high average coulombic efficiency of 99.8%. This work offers a universal strategy for the design of aqueous electrolytes with wide ESW and the ability to effectively suppress vanadium dissolution. [ABSTRACT FROM AUTHOR]

Liquid‒liquid phase separation (LLPS) is a ubiquitous process in which proteins, RNA, and biomolecules assemble into membrane‐less compartments, playing important roles in many biological functions and diseases. The current knowledge on the biophysical and biochemical principles of LLPS is largely from in vitro studies; however, the physiological environment in living cells is complex and not at equilibrium. The characteristics of intracellular dynamics and their roles in physiological LLPS remain to be resolved. Here, by using single‐particle tracking of quantum dots and dynamic monitoring of the formation of stress granules (SGs) in single cells, the spatiotemporal dynamics of intracellular transport in cells undergoing LLPS are quantified. It is shown that intracellular diffusion and active transport are both reduced. Furthermore, the formation of SG droplets contributes to increased spatial heterogeneity within the cell. More importantly, the study demonstrated that the LLPS of SGs can be regulated by intracellular dynamics in two stages: the reduced intracellular diffusion promotes SG assembly and the microtubule‐associated transport facilitates SG coalescences. The work on intracellular dynamics not only improves the understanding of the mechanism of physiology phase separations occurring in nonequilibrium environments but also reveals an interplay between intracellular dynamics and LLPS. [ABSTRACT FROM AUTHOR]

The human genome contains many G-quadruplex-forming sequences, including sequences containing long single-stranded loops that are believed to be unfavorable for G-quadruplex formation. The intracellular environment of biological cells is crowded with proteins with charged surfaces. Understanding the effects of protein-rich environments is important for understanding the formation of G-quadruplexes in an intracellular environment. In this study, we investigated the structural stability of DNA G-quadruplexes in the presence of several types of globular proteins (lysozyme, cytochrome c , bovine serum albumin, myoglobin, histone proteins, and serum proteins), unstructured polypeptides (protamine and poly- l -lysine), and oligopeptides (RGG/RG-domain peptides and short repeated peptides). Thermal melting studies of G-quadruplex-forming oligonucleotides derived from the human telomeric repeat sequence revealed that environments containing high concentrations of proteins and peptides differently affected the G-quadruplex stability according to their loop lengths. We found that weak electrostatic interactions of G-quadruplex loops with basic proteins and peptides improved the stability of long-loop G-quadruplexes and the interactions were strengthened under crowded conditions simulated by dextran. The comparison of the effects of different types of proteins and peptides indicated that excluded volume interactions and structural flexibility of both DNA and polypeptide chains influenced the efficiency of their interactions. This study provides insights into long-loop G-quadruplex stability in a crowded intracellular environment and the recognition of G-quadruplexes by arginine-rich domains of G-quadruplex-binding proteins. [Display omitted] • Basic protein- and peptide-induced improvement of the stability of DNA G-quadruplex. • Weak electrostatic interactions between long-loop G-quadruplexes and basic proteins. • Enhanced interaction of G-quadruplexes with basic proteins under crowded conditions. • Different binding properties depending on flexibility of DNA and polypeptide chains. [ABSTRACT FROM AUTHOR]

The cytoplasm is densely packed with molecules that contribute to its nonideal behavior. Cytosolic crowding influences chemical reaction rates, intracellular water mobility, and macromolecular complex formation. Overcrowding is potentially catastrophic; to counteract this problem, cells have evolved acute and chronic homeostatic mechanisms that optimize cellular crowdedness. Here, we provide a physiology-focused overview of molecular crowding, highlighting contemporary advances in our understanding of its sensing and control. Long hypothesized as a form of crowding-induced microcompartmentation, phase separation allows cells to detect and respond to intracellular crowding through the action of biomolecular condensates, as indicated by recent studies. Growing evidence indicates that crowding is closely tied to cell size and fluid volume, homeostatic responses to physical compression and desiccation, tissue architecture, circadian rhythm, aging, transepithelial transport, and total body electrolyte and water balance. Thus, molecular crowding is a fundamental physiologic parameter that impacts diverse functions extending from molecule to organism. [ABSTRACT FROM AUTHOR]

Abstract Liquid‒liquid phase separation (LLPS) is a ubiquitous process in which proteins, RNA, and biomolecules assemble into membrane‐less compartments, playing important roles in many biological functions and diseases. The current knowledge on the biophysical and biochemical principles of LLPS is largely from in vitro studies; however, the physiological environment in living cells is complex and not at equilibrium. The characteristics of intracellular dynamics and their roles in physiological LLPS remain to be resolved. Here, by using single‐particle tracking of quantum dots and dynamic monitoring of the formation of stress granules (SGs) in single cells, the spatiotemporal dynamics of intracellular transport in cells undergoing LLPS are quantified. It is shown that intracellular diffusion and active transport are both reduced. Furthermore, the formation of SG droplets contributes to increased spatial heterogeneity within the cell. More importantly, the study demonstrated that the LLPS of SGs can be regulated by intracellular dynamics in two stages: the reduced intracellular diffusion promotes SG assembly and the microtubule‐associated transport facilitates SG coalescences. The work on intracellular dynamics not only improves the understanding of the mechanism of physiology phase separations occurring in nonequilibrium environments but also reveals an interplay between intracellular dynamics and LLPS.

The pathological process of prion diseases implicates that the normal physiological cellular prion protein (PrPC) converts into misfolded abnormal scrapie prion (PrPSc) through post-translational modifications that increase β-sheet conformation. We recently demonstrated that HuPrP(90–231) thermal unfolding is partially irreversible and characterized by an intermediate state (β-PrPI), which has been revealed to be involved in the initial stages of PrPC fibrillation, with a seeding activity comparable to that of human infectious prions. In this study, we report the thermal unfolding characterization, in cell-mimicking conditions, of the truncated (HuPrP(90–231)) and full-length (HuPrP(23–231)) human prion protein by means of CD and NMR spectroscopy, revealing that HuPrP(90–231) thermal unfolding is characterized by two successive transitions, as in buffer solution. The amyloidogenic propensity of HuPrP(90–231) under crowded conditions has also been investigated. Our findings show that although the prion intermediate, structurally very similar to β-PrPI, forms at a lower temperature compared to when it is dissolved in buffer solution, in cell-mimicking conditions, the formation of prion fibrils requires a longer incubation time, outlining how molecular crowding influences both the equilibrium states of PrP and its kinetic pathways of folding and aggregation.

The cell interior is a highly crowded 'active material': ATP-consuming, non-equilibrium activities help fluidize the cell interior. Without metabolic energy, the crowded environment can become 'glassy' or solid like. Molecular crowding reduces particle diffusion, especially at the 'mesoscale' (10–1000 nm diameter) but can increase molecular assembly through 'depletion attraction'. We can probe molecular crowding and the active matter of the cell interior by observing the motion of tracer particles. This is called 'microrheology'. Both internal and external factors influence the biophysical properties of the cell interior: for example, decreased target of rapamycin complex 1 (TORC1) signaling reduces crowding, while mechanical compression increases crowding. Active control of these properties is crucial for normal physiology; we hypothesize that perturbed molecular crowding or decreased non-equilibrium motion could contribute to diseases like cancer and neurodegeneration. Life emerges from thousands of biochemical processes occurring within a shared intracellular environment. We have gained deep insights from in vitro reconstitution of isolated biochemical reactions. However, the reaction medium in test tubes is typically simple and diluted. The cell interior is far more complex: macromolecules occupy more than a third of the space, and energy-consuming processes agitate the cell interior. Here, we review how this crowded, active environment impacts the motion and assembly of macromolecules, with an emphasis on mesoscale particles (10–1000 nm diameter). We describe methods to probe and analyze the biophysical properties of cells and highlight how changes in these properties can impact physiology and signaling, and potentially contribute to aging, and diseases, including cancer and neurodegeneration. [ABSTRACT FROM AUTHOR]

Allophycocyanin (APC) is a key molecule of the light harvesting complex - phycobilisome of Cyanobacteria and Red Algae. Its bright color and fluorescent nature make it a suitable molecular for biomedical applications. Here, we report the dilution induced changes in the spectral and structural properties of APC, isolated from marine cyanobacterium, Phormidium sp. A09DM. We observe measurable blue shift in the light absorption and fluorescence emission properties of APC upon dilution. The blue shift in spectral properties is originated from the disruption of hydrophobic interactions and systematic dissociation of the APC into its building blocks. APC, a trimer of αβ-heterodimer, dissociates into monomer and further into individual subunits upon dilution. The observed dilution induced spectral and structural changes can be moderated in the presence of physiological osmolytes with the moderation strength in the order, Sucrose > Trehalose > Betaine = Potassium Phosphate buffer > Glycine. Our results conclude that the spectral properties of APC is a function of osmolytes concentrations, which makes it a potential candidate to be explored as the molecular probe for measuring the molecular crowding. [Display omitted] • Allophycocyanin (APC) exhibits spectral blue shift upon dilution. • APC dissociates into monomers upon dilution. • Structure and spectral properties of APC is a function of molecular crowding. [ABSTRACT FROM AUTHOR]

Molecular crowding environments play a crucial role in understanding the mechanisms of biological reactions. Inside living cells, a diverse array of molecules coexists within a volume fraction ranging from 10% to 30% v/v. However, conventional spectroscopic methods often face difficulties in selectively observing the structures of particular proteins or membranes within such molecularly crowded environments due to the presence of high background signals. Therefore, it is crucial to establish in vitro measurement conditions that closely resemble the intracellular environment. Meanwhile, the neutron scattering method offers a significant advantage in selectively observing target biological components, even within crowded environments. Recently, we have demonstrated a novel scattering method capable of selectively detecting the structures of targeted proteins or membranes in a closely mimicking intracellular milieu achieved utilizing whole-cell contents (deuterated-cell debris). This method relies on the inverse contrast matching technique in neutron scattering. By employing this method, we successfully observed the fibrillization process of human amyloid beta-protein (Aβ 1–40) under a molecular crowding environment (13.1% w/v cell debris, Aβ/cell debris = ~1/25 w/w) that closely mimics the interior of living cells. Aβ protein is well known as a major pathogenic component of Alzheimer's disease. The present results combining model simulation analyses clearly show that the intracellular environment facilitates the potential formation of even more intricate higher-order aggregates of Aβ proteins than those previously reported. [ABSTRACT FROM AUTHOR]

SARS‐CoV‐2 aptamer is a favorable candidate for the recognition and detection of SARS‐CoV‐2, owing to its small size and easy synthesis. However, the issue of compromised binding affinities in real samples and targeting mutant SARS‐CoV‐2 hinder wide applications of the aptamer. In this study, it is discovered that molecular crowding could increase binding affinity of CoV2‐6C3 aptamer against RBD (Receptor Binding Domain) of SARS‐CoV‐2 via increasing the absolute value of the enthalpy change. The values of the equilibrium dissociation constant in molecular crowding decrease by 70% and 150%, respectively, against wild‐type and mutant RBD compared with those in buffer without crowding. Moreover, the detection limit of SARS‐CoV‐2 pseudovirus is up to 5 times lower under molecular crowding compared to dilute conditions. The discovery deepens the understanding of aptamer‐target interaction mechanisms in crowding conditions and provides an effective way to apply SARS‐CoV‐2 aptamer for virus recognition and detection. [ABSTRACT FROM AUTHOR]

SARS‐CoV‐2 aptamer is a favorable candidate for the recognition and detection of SARS‐CoV‐2, owing to its small size and easy synthesis. However, the issue of compromised binding affinities in real samples and targeting mutant SARS‐CoV‐2 hinder wide applications of the aptamer. In this study, it is discovered that molecular crowding could increase binding affinity of CoV2‐6C3 aptamer against RBD (Receptor Binding Domain) of SARS‐CoV‐2 via increasing the absolute value of the enthalpy change. The values of the equilibrium dissociation constant in molecular crowding decrease by 70% and 150%, respectively, against wild‐type and mutant RBD compared with those in buffer without crowding. Moreover, the detection limit of SARS‐CoV‐2 pseudovirus is up to 5 times lower under molecular crowding compared to dilute conditions. The discovery deepens the understanding of aptamer‐target interaction mechanisms in crowding conditions and provides an effective way to apply SARS‐CoV‐2 aptamer for virus recognition and detection.

Optically active liquid–crystalline dispersions (LCD) of nucleic acids, obtained by polymer- and salt-induced (psi-) condensation, e.g., by mixing of aqueous saline solutions of low molecular weight DNA (≤106 Da) and polyethylene glycol (PEG), possess an outstanding circular dichroism (CD) signal (so-called psi-CD) and are of interest for sensor applications. Typically, such CD signals are observed in PEG content from ≈12.5% to ≈22%. However, in the literature, there are very conflicting data on the existence of psi-CD in DNA LCDs at a higher content of crowding polymer up to 30–40%. In the present work, we demonstrate that, in the range of PEG content in the system above ≈24%, optically polymorphic LCDs can be formed, characterized by both negative and positive psi-CD signals, as well as by ones rather slightly differing from the spectrum of isotropic DNA solution. Such a change in the CD signal is determined by the concentration of the stock solution of PEG used for the preparation of LCDs. We assume that various saturation of polymer chains with water molecules may affect the amount of active water, which in turn leads to a change in the hydration of DNA molecules and their transition from B-form to Z-form. [ABSTRACT FROM AUTHOR]

In order to investigate the effect of molecular crowding on the antifungal ability of DNA-AgNCs and its preservation effect on citrus, DNA sequence (5'-CCCCCCCCCCCC-3') was mixed with AgNO3 solution in this experiment. DNA-AgNCs was prepared by simulating molecular crowded environment with 50% Polyethylene Glycol (PEG) 200 solution. Taking DNA-AgNCs as the research object, using Penicillium expansum, Aspergillus flavus, Fusarium graminaerum and Fusarium moniliforme as the models, the inhibition rate of DNA-AgNCs on the germination of four kinds of spores was determined by spore counting method, and the Minimal Inhibitory Concentration (MIC) of DNA-AgNCs against four kinds of fungi was determined by microdilution method. DNA-AgNCs was added to starch-chitosan matrix to prepare composite films, and its effects on the mechanical properties, hydrophobicity and antibacterial properties of the films were explored through tensile test, water contact angle and inhibition zone test. The film was applied to citrus preservation in the form of coating film, and the weight loss rate, plaque diameter and sensory evaluation of citrus were recorded. The results showed that the inhibitory effect of molecular crowded DNA-AgNCs on spore germination was better than that of normal DNA-AgNCs (P

Two isoforms of β‐amyloid peptides, Aβ40 and Aβ42, differ from each other only in the last two amino acids, IA, at the end of Aβ42. They, however, differ significantly in their ability in inducing Alzheimer's disease (AD). The rate curves of fibril growth of Aβ40 and Aβ42 and the effects of molecular crowding have been measured in in vitro experiments. These experimental curves, on the other hand, have been fitted in terms of rate constants for elementary reaction steps using rate equation approaches. Several sets of such rate parameters have been reported in the literature. Employing a recently developed stochastic kinetic method, implemented in a browser‐based simulator, popsim, we study to reveal the differences in the kinetic behaviors implied by these sets of rate parameters. In particular, the stochastic method is used to distinguish the kinetic behaviors between Aβ40 and Aβ42 isoforms. As a result, we make general comments on the usefulness of these sets of rate parameters. [ABSTRACT FROM AUTHOR]

ABSTRACT Faster-growing cells must synthesize proteins more quickly. Increased ribosome abundance only partly accounts for increases in total protein synthesis rates. The productivity of individual ribosomes must increase too, almost doubling by an unknown mechanism. Prior models point to diffusive transport as a limiting factor but raise a paradox: faster-growing cells are more crowded, yet crowding slows diffusion. We suspected that physical crowding, transport, and stoichiometry, considered together, might reveal a more nuanced explanation. To investigate, we built a first-principles physics-based model of Escherichia coli cytoplasm in which Brownian motion and diffusion arise directly from physical interactions between individual molecules of finite size, density, and physiological abundance. Using our microscopically detailed model, we predicted that physical transport of individual ternary complexes accounts for ~80% of translation elongation latency. We also found that volumetric crowding increases during faster growth even as cytoplasmic mass density remains relatively constant. Despite slowed diffusion, we predicted that improved proximity between ternary complexes and ribosomes wins out, illustrating a simple physics-based mechanism for how individual elongating ribosomes become more productive. We speculate that crowding imposes a physical limit on growth rate and undergirds cellular behavior more broadly. Unfitted colloidal-scale modeling offers systems biology a complementary “physics engine” for exploring how cellular-scale behaviors arise from physical transport and reactions among individual molecules. IMPORTANCE Ribosomes are the factories in cells that synthesize proteins. When cells grow faster, there are not enough ribosomes to keep up with the demand for faster protein synthesis without individual ribosomes becoming more productive. Yet, faster-growing cells are more crowded, seemingly making it harder for each ribosome to do its work. Our computational model of the physics of translation elongation reveals the underlying mechanism for how individual ribosomes become more productive: proximity and stoichiometry of translation molecules overcome crowding. Our model also suggests a universal physical limitation of cell growth rates.

Although biophysical studies have traditionally been performed in diluted solutions, it was pointed out in the late 1990s that the cellular milieu contains several other macromolecules, creating a condition of molecular crowding. How crowding affects protein stability is an important question heatedly discussed over the past 20 years. Theoretical estimations have suggested a 5–20°C effect of fold stabilisation. This estimate, however, is at variance with what has been verified experimentally that proposes only a limited increase of stability, opening the question whether some of the assumptions taken for granted should be reconsidered. The present review critically analyses the causes of this discrepancy and discusses the limitations and implications of the current concept of crowding. The interior of cells is an aqueous solution much more concentrated than buffers used for in vitro studies, posing the problem of how such 'crowded' conditions influence protein stability and other properties. The presence of high concentrations of crowders limits the volume available to proteins under study and impacts their thermal stability. The influence of crowders on protein stability is modulated by the presence of enthalpic contributions, which may shield the real mechanism of crowding. Careful studies that minimise weak interactions support the importance of entropic contributions. [ABSTRACT FROM AUTHOR]

Molecular crowding environments play a crucial role in understanding the mechanisms of biological reactions. Inside living cells, a diverse array of molecules coexists within a volume fraction ranging from 10% to 30% v/v. However, conventional spectroscopic methods often face difficulties in selectively observing the structures of particular proteins or membranes within such molecularly crowded environments due to the presence of high background signals. Therefore, it is crucial to establish in vitro measurement conditions that closely resemble the intracellular environment. Meanwhile, the neutron scattering method offers a significant advantage in selectively observing target biological components, even within crowded environments. Recently, we have demonstrated a novel scattering method capable of selectively detecting the structures of targeted proteins or membranes in a closely mimicking intracellular milieu achieved utilizing whole-cell contents (deuterated-cell debris). This method relies on the inverse contrast matching technique in neutron scattering. By employing this method, we successfully observed the fibrillization process of human amyloid beta-protein (Aβ 1–40) under a molecular crowding environment (13.1% w/v cell debris, Aβ/cell debris = ~1/25 w/w) that closely mimics the interior of living cells. Aβ protein is well known as a major pathogenic component of Alzheimer’s disease. The present results combining model simulation analyses clearly show that the intracellular environment facilitates the potential formation of even more intricate higher-order aggregates of Aβ proteins than those previously reported.

The cytoophidium is a unique type of membraneless compartment comprising of filamentous protein polymers. Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step of de novo GTP biosynthesis and plays critical roles in active cell metabolism. However, the molecular regulation of cytoophidium formation is poorly understood. Here we show that human IMPDH2 polymers bundle up to form cytoophidium-like aggregates in vitro when macromolecular crowders are present. The self-association of IMPDH polymers is suggested to rely on electrostatic interactions. In cells, the increase of molecular crowding with hyperosmotic medium induces cytoophidia, while the decrease of that by the inhibition of RNA synthesis perturbs cytoophidium assembly. In addition to IMPDH, CTPS and PRPS cytoophidium could be also induced by hyperosmolality, suggesting a universal phenomenon of cytoophidium-forming proteins. Finally, our results indicate that the cytoophidium can prolong the half-life of IMPDH, which is proposed to be one of conserved functions of this subcellular compartment. [ABSTRACT FROM AUTHOR]

Point of care (PoC) nucleic acid amplification tests (NAATs) are a cornerstone of public health, providing the earliest and most accurate diagnostic method for many communicable diseases in the same location where the patient receives treatment. Communicable diseases, such as human immunodeficiency virus (HIV), disproportionately impact low-resource communities where NAATs are often unobtainable due to the resource-intensive enzymes that drive the tests. Enzyme-free nucleic acid detection methods, such as hybridization chain reaction (HCR), use DNA secondary structures for self-driven amplification schemes, producing large DNA nanostructures, capable of single-molecule detection in cellulo . These thermodynamically driven DNA-based tests have struggled to penetrate the PoC diagnostic field due to their inadequate limits of detection or complex workflows. Here, we present a proof-of-concept NAAT that combines HCR-based amplification of a target nucleic acid sequence with paper-based nucleic acid filtration and enrichment capable of detecting sub-pM levels of synthetic DNA. We reconstruct the favorable hybridization conditions of an in cellulo reaction in vitro by incubating HCR in an evaporating, microvolume environment containing poly(ethylene glycol) as a crowding agent. We demonstrate that the kinetics and thermodynamics of DNA-DNA and DNA-RNA hybridization is enhanced by the dynamic evaporating environment and inclusion of crowding agents, bringing HCR closer to meeting PoC NAAT needs.

The class of intrinsically disordered proteins lacks stable three-dimensional structures. Their flexibility allows them to engage in a wide variety of interactions with other biomolecules thus making them biologically relevant and efficient. The intrinsic disorders of these proteins, which undergo binding-induced folding, allow alterations in their topologies while conserving their binding sites. Due to the lack of well-defined three-dimensional structures in the absence of their physiological partners, the folding and the conformational dynamics of these proteins remained poorly understood. Particularly, it is unclear how these proteins exist in the crowded intracellular milieu. In the present study, molecular dynamic simulations of two intrinsically unstructured proteins and two controls (folded proteins) were conducted in the presence and absence of molecular crowders to obtain an in-depth insight into their conformational flexibility. The present study revealed that polymer crowders stabilize the disordered proteins through enthalpic as well as entropic effects that are significantly more than their monomeric counterpart. Taken together, the study delves deep into crowding effects on intrinsically disordered proteins and provides insights into how molecular crowders induce a significantly diverse ensemble of dynamic scaffolds needed to carry out diverse functions.Communicated by Ramaswamy H. Sarma.

Lipid vesicles are valuable mesoscale molecular confinement vessels for studying membrane mechanics and lipid–protein interactions, and they have found utility among bio-inspired technologies, including drug delivery vehicles. While vesicle morphology can be modified by changing the lipid composition and introducing fusion or pore-forming proteins and detergents, the influence of extramembrane crowding on vesicle morphology has remained under-explored owing to a lack of experimental tools capable of capturing morphological changes on the nanoscale. Here, we use biocompatible polymers to simulate molecular crowding in vitro, and through combinations of FRET spectroscopy, lifetime analysis, dynamic light scattering, and single-vesicle imaging, we characterize how crowding regulates vesicle morphology. We show that both freely diffusing and surface-tethered vesicles fluorescently tagged with the DiI and DiD FRET pair undergo compaction in response to modest concentrations of sorbitol, polyethylene glycol, and Ficoll. A striking observation is that sorbitol results in irreversible compaction, whereas the influence of high molecular weight PEG-based crowders was found to be reversible. Regulation of molecular crowding allows for precise control of the vesicle architecture in vitro, with vast implications for drug delivery and vesicle trafficking systems. Furthermore, our observations of vesicle compaction may also serve to act as a mechanosensitive readout of extramembrane crowding.

Abstract Solid surfaces that are immobilized with DNA molecules underlie an array of biotechnological devices. These surfaces may also mediate the self‐assembly of hierarchical DNA nanostructures. However, a number of fundamental questions concerning the structure–function relationship of these biointerfaces remain, including how these DNA probe molecules organize on the surface and how the spatial organization influences molecular recognition kinetics and interfacial affinity of these DNA molecules at the regime where crowding interactions are important (1–10 nm). This mini‐review covers recent advances in understanding this structure–function relationship by spatially resolving surface hybridization events at the single‐molecule level. Counterintuitive cooperative effects in surface hybridization are discussed and as is how modeling these cooperative effects can be used to predict the hybridization kinetics of a prototypical DNA sensor. Future opportunities in using mechanistic understanding to improve the performance and reliability of DNA sensors and form hierarchical supramolecular structures are also discussed.

Biopolymer networks play a major role as part of the cytoskeleton. They provide stable structures and act as a medium for signal transport. These features encourage the application of such networks as organic computation devices. While research on this topic is not advanced yet, previous results are very promising. The protein actin in particular appears advantageous. It can be arranged to various stable structures and transmit several signals. In this study aster shaped networks were self-assembled via entropic forces by the crowding agent methyl cellulose. These networks are characterised by a regular and uniquely thick bundle structure, but have so far only been accounted in droplets of 100 μm diameter. We report now regular asters in an area of a few mm2 that could be observed even after months. Such stability outside of an organism is striking and underlines the great potential actin aster networks display.

Optically active liquid–crystalline dispersions (LCD) of nucleic acids, obtained by polymer- and salt-induced (psi-) condensation, e.g., by mixing of aqueous saline solutions of low molecular weight DNA (≤106 Da) and polyethylene glycol (PEG), possess an outstanding circular dichroism (CD) signal (so-called psi-CD) and are of interest for sensor applications. Typically, such CD signals are observed in PEG content from ≈12.5% to ≈22%. However, in the literature, there are very conflicting data on the existence of psi-CD in DNA LCDs at a higher content of crowding polymer up to 30–40%. In the present work, we demonstrate that, in the range of PEG content in the system above ≈24%, optically polymorphic LCDs can be formed, characterized by both negative and positive psi-CD signals, as well as by ones rather slightly differing from the spectrum of isotropic DNA solution. Such a change in the CD signal is determined by the concentration of the stock solution of PEG used for the preparation of LCDs. We assume that various saturation of polymer chains with water molecules may affect the amount of active water, which in turn leads to a change in the hydration of DNA molecules and their transition from B-form to Z-form.

Protein amyloid fibrils have widespread implications for human health. Over the last twenty years, fibrillation has been studied using a variety of crowding agents to mimic the packed interior of cells or to probe the mechanisms and pathways of the process. We tabulate and review these results by considering three classes of crowding agent: synthetic polymers, osmolytes and other small molecules, and globular proteins. While some patterns are observable for certain crowding agents, the results are highly variable and often depend on the specific pairing of crowder and fibrillating protein. [ABSTRACT FROM AUTHOR]

Given the biochemical reaction stochasticity, the mechanisms leading to conservation of biological functions from noise are obscure. Pervasive transcription of nonconserved genomic regions generates lowly expressed cancer-specific long noncoding RNAs (lncRNAs). How such poorly expressed transcripts, often undetectable in normal tissues, consistently modulate the activity of multiple abundant proteins leading to cancer phenotypes is unclear. Biochemical reaction compartmentalisation in response to environmental oscillations through liquid–liquid phase separation (LLPS) may explain the emergence of order from molecular noise. LncRNAs contain repetitive sequences and as such contribute to molecular crowding and LLPS. We propose that lncRNAs mediate cancer stress signals by regulating aberrant LLPS. This emerging model and its consequences for stoichiometry and specificity may lead to the development of diagnostic tools and cancer-specific drugs. The human (cancer) genome is pervasively transcribed into a plethora of noncoding transcripts that are mostly not conserved, lowly expressed, and consistently derepressed in cancer. Long noncoding RNAs (lncRNAs) exert multiple key molecular functions in cancer, converging towards the regulation of epigenetic and post- transcriptional events. The lack of conservation and the low expression of lncRNAs are in striking contrast with their key role in all epigenetic and post-transcriptional processes in the cell, thus exposing the CC (conservation and concentration) paradox. LncRNAs are able to drive aberrant liquid–liquid phase separation in cancer in response to stressors. A better understanding of lncRNA-driven cancer aberrant compartmentalisation may lead to the development of new diagnostic and therapeutic tools and to the re-evaluation of basic concepts in RNA–protein interactions. [ABSTRACT FROM AUTHOR]

The behavior of biomolecules in crowded environments remains largely unknown due to the accuracy of simulation models and the limited experimental data for comparison. Here we chose a small crowder of tetraethylene glycol (PEG-4) to investigate the self-crowding of PEG-4 solutions and molecular crowding effects on the structure and diffusion of lysozyme at varied concentrations from dilute water to pure PEG-4 liquid. Two Amber-like force fields of Amber14SB and a99SB-disp were examined with TIP3P (fast diffusivity and low viscosity) and a99SB-disp (slow diffusivity and high viscosity) water models, respectively. Compared to the Amber14SB protein simulations, the a99SB-disp model yields more coordinated water and less PEG-4 molecules, less intramolecular hydrogen bonds (HBs), more protein–water HBs, and less protein–PEG HBs as well as stronger interactions and more hydrophilic and less hydrophobic contacts with solvent molecules. The a99SB-disp model offers comparable protein–solvent interactions in concentrated PEG-4 solutions to that in pure water. The PEG-4 crowding leads to a slow-down in the diffusivity of water, PEG-4, and protein, and the decline in the diffusion from atomistic simulations is close to or faster than the hard sphere model that neglects attractive interactions. Despite these differences, the overall structure of lysozyme appears to be maintained well at different PEG-4 concentrations for both force fields, except a slightly large deviation at 370 K at low concentrations with the a99SB-disp model. This is mainly attributed to the strong intramolecular interactions of the protein in the Amber14SB force field and to the large viscosity of the a99SB-disp water model. The results indicate that the protein force fields and the viscosity of crowder solutions affect the simulation of biomolecules under crowding conditions. [ABSTRACT FROM AUTHOR]

The action of three types of co-solutes: (i) salts (NaCl, NaBr, NaI), (ii) polymer (polyethylene glycol; PEG-400, PEG-3000, PEG-20000), and (iii) sugars (sucrose, sucralose) on the complexation between bovine serum albumin (BSA) and sodium polystyrene sulfonate (NaPSS) was studied. Three critical pH parameters were extracted from the pH dependence of the solution's turbidity: p H c corresponding to the formation of the soluble complexes, p H Φ corresponding to the formation of the insoluble complexes, and p H opt corresponding to the charge neutralization of the complexes. In the presence of salts, the formation of soluble and insoluble complexes as well as the charge neutralization of complexes was hindered, which is a consequence of the electrostatic screening of attractive interactions between BSA and NaPSS. Distinct anion-specific trends were observed in which the stabilizing effect of the salt increased in the order: NaCl < NaBr < NaI. The presence of PEG, regardless of its molecular weight, showed no measurable effect on the formation of soluble complexes. PEG-400 and PEG-3000 showed no effect on the formation of insoluble complexes, but PEG-20000 in high concentrations promoted their formation due to the molecular crowding effect. The presence of sugar molecules had little effect on BSA-NaPSS complexation. Sucralose showed a minor stabilizing effect with respect to the onset of complex formation, which was due to its propensity to the protein surface. This was confirmed by the fluorescence quenching assay (Stern-Volmer relationship) and all-atom MD simulations. This study highlights that when evaluating the modulatory effect of co-solutes on protein-polyelectrolyte interactions, (co-solute)-protein interactions and their subsequent impact on protein aggregation must also be considered. [ABSTRACT FROM AUTHOR]

Phosphoryl guanidine oligonucleotides (PGOs) are promising uncharged analogs of nucleic acids and are used in a variety of applications. The importance of hydration is frequently ignored during the design of modified nucleic acid probes. Such hydrophobic modifications (phosphoryl guanidine) are expected to have a significant impact on the structure and thermal stability of the affected oligo with complementary nucleic acids. Here we aimed to investigate (by the osmotic stress method) hydration changes upon the formation of a duplex of a PGO with complementary DNA. According to our results, the presence of phosphoryl guanidines in one or both strands of a duplex only minimally affects hydration alterations under crowding conditions. The secondary structure of native and modified duplexes did not change significantly in the presence of ethanol, ethylene glycol, polyethylene glycol 200, or polyethylene glycol 1000. After the addition of a cosolvent, the thermodynamic stability of the PGO complexes changed in the same manner as that seen in a corresponding DNA duplex. The findings reported here and our previous studies form the basis for efficient use of PGOs in basic research and a variety of applications. • Cosolvents did not perturb the secondary structure of PGO/DNA duplexes. • A number of released water molecules at formation of DNA or PGO duplex are very close. • Crowding conditions affect on DNA/DNA and DNA/PGO thermodynamics in the same way. • Uncharged PGOs are useful model in basic research and a variety of applications. [ABSTRACT FROM AUTHOR]

• FRET-based crowding sensor used in yeast in high- and low-stress conditions. • Confocal microscopy used to generate subcellular crowding maps. • Yeast experience different cytosolic crowding under different glucose concentrations during growth. • Individual sensor trajectories may be found paving the way for sensor readouts correlated with probe spatiotemporal position. The physical and chemical environment inside cells is of fundamental importance to all life but has traditionally been difficult to determine on a subcellular basis. Here we combine cutting-edge genomically integrated FRET biosensing to readout localized molecular crowding in single live yeast cells. Confocal microscopy allows us to build subcellular crowding heatmaps using ratiometric FRET, while whole-cell analysis demonstrates crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicate that the cell membrane is largely inaccessible to these sensors and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds. Millisecond single-molecule optical microscopy was used to track molecules and obtain brightness estimates that enabled calculation of crowding sensor copy numbers. The quantification of diffusing molecule trajectories paves the way for correlating subcellular processes and the physicochemical environment of cells under stress. [ABSTRACT FROM AUTHOR]

While current group I ribozymes use several distinct strategies to function under conditions of low Mg2+ concentration (≤ 3 mM), a deletion mutant of the Tetrahymena ribozyme (ΔP5 ribozyme) is virtually inactive with 3 mM Mg2+ due to removal of the large peripheral module, P5abc, supporting the active conformation of the core module. We investigated the molecular crowding effects of synthetic polyethylene glycols (PEGs) on the activity of the ΔP5 ribozyme. Among PEG molecules with different chain lengths, PEG600 improved the activity of the ΔP5 ribozyme most effectively in the presence of 3 mM Mg2+. [ABSTRACT FROM AUTHOR]

The cell is three-dimensionally and dynamically organized into cellular compartments, including the endoplasmic reticulum, mitochondria, vesicles, and nucleus, which have high relative molecular density. The structure and functions of these compartments and organelles may be deduced from the diffusion and interaction of related biomolecules. Among these cellular components, various protein molecules can freely access the nucleolus or mitotic chromosome through Brownian diffusion, even though they have a densely packed structure. However, physicochemical properties of the nucleolus and chromosomes, such as molecular density and volume, are not yet fully understood under changing cellular conditions. Many studies have been conducted based on high-resolution imaging and analysis techniques using fluorescence. However, there are limitations in imaging only fluorescently labeled molecules, and cytotoxicity occurs during three-dimensional imaging. Alternatively, the recently developed label-free three-dimensional optical diffraction tomography (ODT) imaging technique can divide various organelles in cells into volumes and analyze them by refractive index, although specific molecules cannot be observed. A previous study established an analytical method that provides comprehensive insights into the physical properties of the nucleolus and mitotic chromosome by utilizing the advantages of ODT and fluorescence techniques, such as fluorescence correlation spectroscopy and confocal laser scanning microscopy. This review article summarizes a recent study and discusses the future aspects of the ODT for cellular compartments.

At dawn of a scorching summer day, land plants must anticipate upcoming extreme midday temperatures by timely establishing molecular defences that can keep heat‐labile membranes and proteins functional. A gradual morning pre‐exposure to increasing sub‐damaging temperatures induces heat‐shock proteins (HSPs) that are central to the onset of plant acquired thermotolerance (AT). To gain knowledge on the mechanisms of AT in the model land plant Physcomitrium patens, we used label‐free LC–MS/MS proteomics to quantify the accumulated and depleted proteins before and following a mild heat‐priming treatment. High protein crowding is thought to promote protein aggregation, whereas molecular chaperones prevent and actively revert aggregation. Yet, we found that heat priming (HP) did not accumulate HSP chaperones in chloroplasts, although protein crowding was six times higher than in the cytosol. In contrast, several HSP20s strongly accumulated in the cytosol, yet contributing merely 4% of the net mass increase of heat‐accumulated proteins. This is in poor concordance with their presumed role at preventing the aggregation of heat‐labile proteins. The data suggests that under mild HP unlikely to affect protein stability. Accumulating HSP20s leading to AT, regulate the activity of rare and specific signalling proteins, thereby preventing cell death under noxious heat stress. Quantitative proteomics showed that a mild heat pre‐treatment leading to acquired thermotolerance in moss strongly induces the accumulation of cytosolic HSP20s, albeit in low amounts compared to other HSPs. The cytosol was found to be the least crowded cellular compartment, suggesting that in addition to their general anti‐aggregation function, cytosolic HSP20s carry specific signalling functions to regulate noxious heat‐induced apoptosis. [ABSTRACT FROM AUTHOR]

Luminescence spectroscopy coupled with molecular rotors was used in the TNO Intestinal Model-1 (TIM-1) to monitor in situ changes to luminal viscosity of three maize starch samples varying in the amylose-to-amylopectin ratio (AM: AP): normal, high amylose (AM) and high amylopectin (AP). The fluorescence intensity (FI) of Fast Green (FG), a proven micro (and bulk) viscosity probe, was monitored throughout digestion to track changes in the gastric viscosity. The FI of FG and the viscosity imparted by the starch followed a power-law relationship. The emission of the MR was unaffected by the composition of TIM-1 secretion fluids nor pH. Hence, direct measurements of digesta FI are sensitive to changing viscosity during the simulated digestion. The viscosity was highest for AP, followed by normal starch, and high AM had the lowest viscosity. In the TIM-1 gastric compartment, from highest to lowest FI, and thus viscosity was high AM > high AP > normal maize starches. We conclude the validity of the proposed method to facilitate the measurement of luminal viscosity, in vitro, when the microviscosity represents bulk viscosity (i.e., when the increase in bulk viscosity is a result of molecular crowding and the surrounding environment around the rotor is homogeneous). Careful consideration is required when foods are heterogeneous as molecular rotors report only on their local non-uniform environment. [ABSTRACT FROM AUTHOR]

The right-handed double-helical B-form structure (B-form duplex) has been widely recognized as the canonical structure of nucleic acids since it was first proposed by James Watson and Francis Crick in 1953. This B-form duplex model has a monochronic and static structure and codes genetic information within a sequence. Interestingly, DNA and RNA can form various non-canonical structures, such as hairpin loops, left-handed helices, triplexes, tetraplexes of G-quadruplex and i-motif, and branched junctions, in addition to the canonical structure. The formation of non-canonical structures depends not only on sequence but also on the surrounding environment. Importantly, these non-canonical structures may exhibit a wide variety of biological roles by changing their structures and stabilities in response to the surrounding environments, which undergo vast changes at specific locations and at specific times in cells. Here, we review recent progress regarding the interesting behaviors and functions of nucleic acids controlled by molecularly crowded cellular conditions. New insights gained from recent studies suggest that nucleic acids not only code genetic information in sequences but also have unknown functions regarding their structures and stabilities through drastic structural changes in cellular environments. [ABSTRACT FROM AUTHOR]

RNA molecules with trinucleotide repeat expansions are involved in the pathology of various neurodegenerative diseases, such as Huntington's disease. A central research objective is to understand how trinucleotide repeat RNAs fold and function in disease progression. Studies that investigate the sequence structure, stability and self‐assembly of such RNAs mainly focused on in vitro methods in dilute aqueous solution. Functional studies in cells reveal further aspects of transport, protein association or assembly in phase separated compartments. Our study aims to understand governing factors for folding stability of trinucleotide repeats on the cellular level by assessing the influence of key components of the cellular medium in vitro. We investigate (CAG)20 RNA in complex and cytomimetic media using Fast Relaxation Imaging. Using distinct cosolutes we analyze the different enthalpic and entropic contributions that determine folding stability. We find a remarkable destabilization and aggregation of the RNA in cellular lysate that can be attributed to specific and non‐specific interactions with the unfolded state. [ABSTRACT FROM AUTHOR]

Protein amyloid fibrils have widespread implications for human health. Over the last twenty years, fibrillation has been studied using a variety of crowding agents to mimic the packed interior of cells or to probe the mechanisms and pathways of the process. We tabulate and review these results by considering three classes of crowding agent: synthetic polymers, osmolytes and other small molecules, and globular proteins. While some patterns are observable for certain crowding agents, the results are highly variable and often depend on the specific pairing of crowder and fibrillating protein.

In the last decades, it has become increasingly clear how the modulation of spatial organization of chromatin over time and through the cell cycle is closely connected to gene function regulation. Different physicochemical stimuli contribute to the realization of specific transcriptional programs and finally to a specific cellular phenotype. In this review, we aim to describe the current knowledge about the dynamics regulating the movements and the interactions of molecules within the nucleus and their impact on gene functions. In particular, taking into account that these forces exert their effect in a nuclear environment characterized by a high concentration of molecules, we will discuss the role of proteins and structures that regulate these movements and transduce physicochemical signals acting on the cell to the nucleus. [ABSTRACT FROM AUTHOR]

Summary: The nuclear pore complex forms a highly crowded selective barrier with intrinsically disordered regions at the nuclear membrane to coordinate nucleocytoplasmic molecular communications. Although oxidative stress is known to alter the barrier function, the molecular mechanism underlying this adaptive control of the nuclear pore complex remains unknown. Here we uncover a systematic control of the crowding barrier within the nuclear pore in response to various redox environments. Direct measurements of the crowding states using a crowding-sensitive FRET (Förster resonance energy transfer) probe reveal specific roles of the nuclear pore subunits that adjust the degree of crowding in response to different redox conditions, by adaptively forming or disrupting redox-sensitive disulfide bonds. Relationships between crowding control and the barrier function of the nuclear pore are investigated by single-molecular fluorescence measurements of nuclear transport. Based on these findings, we propose a proximal control model of molecular crowding in vivo that is dynamically regulated at the molecular level.

The action of three types of co-solutes: (i) salts (NaCl, NaBr, NaI), (ii) polymer (polyethylene glycol; PEG-400, PEG-3000, PEG-20000), and (iii) sugars (sucrose, sucralose) on the complexation between bovine serum albumin (BSA) and sodium polystyrene sulfonate (NaPSS) was studied. Three critical pH parameters were extracted from the pH dependence of the solution’s turbidity: pHc corresponding to the formation of the soluble complexes, pHΦ corresponding to the formation of the insoluble complexes, and pHopt corresponding to the charge neutralization of the complexes. In the presence of salts, the formation of soluble and insoluble complexes as well as the charge neutralization of complexes was hindered, which is a consequence of the electrostatic screening of attractive interactions between BSA and NaPSS. Distinct anion-specific trends were observed in which the stabilizing effect of the salt increased in the order: NaCl < NaBr < NaI. The presence of PEG, regardless of its molecular weight, showed no measurable effect on the formation of soluble complexes. PEG-400 and PEG-3000 showed no effect on the formation of insoluble complexes, but PEG-20000 in high concentrations promoted their formation due to the molecular crowding effect. The presence of sugar molecules had little effect on BSA-NaPSS complexation. Sucralose showed a minor stabilizing effect with respect to the onset of complex formation, which was due to its propensity to the protein surface. This was confirmed by the fluorescence quenching assay (Stern-Volmer relationship) and all-atom MD simulations. This study highlights that when evaluating the modulatory effect of co-solutes on protein-polyelectrolyte interactions, (co-solute)-protein interactions and their subsequent impact on protein aggregation must also be considered.

The behavior of biomolecules in crowded environments remains largely unknown due to the accuracy of simulation models and the limited experimental data for comparison. Here we chose a small crowder of tetraethylene glycol (PEG-4) to investigate the self-crowding of PEG-4 solutions and molecular crowding effects on the structure and diffusion of lysozyme at varied concentrations from dilute water to pure PEG-4 liquid. Two Amber-like force fields of Amber14SB and a99SB-disp were examined with TIP3P (fast diffusivity and low viscosity) and a99SB-disp (slow diffusivity and high viscosity) water models, respectively. Compared to the Amber14SB protein simulations, the a99SB-disp model yields more coordinated water and less PEG-4 molecules, less intramolecular hydrogen bonds (HBs), more protein–water HBs, and less protein–PEG HBs as well as stronger interactions and more hydrophilic and less hydrophobic contacts with solvent molecules. The a99SB-disp model offers comparable protein–solvent interactions in concentrated PEG-4 solutions to that in pure water. The PEG-4 crowding leads to a slow-down in the diffusivity of water, PEG-4, and protein, and the decline in the diffusion from atomistic simulations is close to or faster than the hard sphere model that neglects attractive interactions. Despite these differences, the overall structure of lysozyme appears to be maintained well at different PEG-4 concentrations for both force fields, except a slightly large deviation at 370 K at low concentrations with the a99SB-disp model. This is mainly attributed to the strong intramolecular interactions of the protein in the Amber14SB force field and to the large viscosity of the a99SB-disp water model. The results indicate that the protein force fields and the viscosity of crowder solutions affect the simulation of biomolecules under crowding conditions.

The perpetually changing cellular conditions, nucleotide sequence, and environmental effects including osmotic stress have multiple effects on DNA, leading to several conformational alternations and subsequently influencing their activity, too. In this work, single-molecule FRET microspectroscopy has been employed to monitor the breathing dynamics as an effect of molecular crowding in the stem region of Fork-DNA. The structural integrity greatly alters with the presence or absence of nucleotide overhangs and on the nature and concentration of the crowding agent, thus affecting the stability of the stem region and hence the forked DNA. The multiple hydrogen bonds and hydrophobic interactions between the polynucleotide strands appear to be altered with osmotic crowding. This induces increased flexibility in the double helix and allows DNA to breath. The conformational alternation of the DNA happens in nanometer resolution, that is been monitored by the change in the FRET efficiency between the dyes attached to two different strands of the DNA. The nature and molecular weight of crowding agents control the degree of spatial breathing in the stem of Fork-DNA. These constant fluctuations between the entropically favorable partially folded structures to an enthalpically favorable folded structure are not only valuable for elucidating nucleic acid structure but might play an important role in enzyme kinetics. Unlabelled Image • Single-molecule FRET has been employed to study breathing in the stem of Fork-DNA. • The role of concentration and size of molecular crowder has been elaborated. • Detailed kinetics using HMM and free energy of each state of fluctuating Fork-DNA has been calculated, • Enthalpically and entropically favorable states have been identified. [ABSTRACT FROM AUTHOR]