Crowder Concentration Research Articles

Comparing effects of attractive interactions in crowded systems: nonspecific, hydrophobic, and hydrogen bond interactions

The equilibrium stability of a protein is determined by its amino acid sequence and the solution conditions, such as temperature, pH and presence of chemical denaturant. The stability of a single protein in two identical solutions can nonetheless differ if other macromolecules, termed cosolutes or crowders, are present in one of the solutions at concentrations high enough to occupy a substantial fraction of the solution volume. This effect, due to the presence of the crowders, decreases or increases the stability depending on the interactions between the protein and crowders. Hard-core steric repulsions, which are responsible for the reduction in free volume, are expected to entropically stabilize the protein while attractive interactions can be destabilizing. Here we use a coarse-grained protein model to assess the impact of different types of crowder-protein interactions on the stability of a 35-amino acid model sequence folding into a helical bundle. We find that, for the same interaction strength and concentration, spherical crowders with a hydrophobic character are more destabilizing than crowders interacting nonspecifically with the protein. However, the two types of interactions differ in the degree of association between crowders and protein. At an interaction strength for which the attractive interactions roughly counteracts the stabilizing hard-core repulsions, the nonspecific interactions lead to much stronger crowder-protein association than the hydrophobic interactions. Additionally, we study crowders in the form of polypeptide chains, which are capable of hydrogen bonding with the protein. These peptide crowders have a destabilizing effect even at relatively low crowder concentrations, especially if the sequence of the peptide crowders includes hydrophobic amino acids. Our findings emphasize the importance of the interplay between different types of attractive crowder-protein interactions and entropic effects in determining the net effect on protein stability.

What determines sub-diffusive behavior in crowded protein solutions?

The aqueous environment inside cells is densely packed. A typical cell has a macromolecular concentration in the range 90–450 g/L, with 5%–40% of its volume being occupied by macromolecules, resulting in what is known as macromolecular crowding. The space available for the free diffusion of metabolites and other macromolecules is thus greatly reduced, leading to so-called excluded volume effects. The slow diffusion of macromolecules under crowded conditions has been explained using transient complex formation. However, sub-diffusion noted in earlier works is not well characterized, particularly the role played by transient complex formation and excluded volume effects. We have used Brownian dynamics simulations to characterize the diffusion of chymotrypsin inhibitor 2 in protein solutions of bovine serum albumin and lysozyme at concentrations ranging from 50 to 300 g/L. The predicted changes in diffusion coefficient as a function of crowder concentration are consistent with NMR experiments. The sub-diffusive behavior observed in the sub-microsecond timescale can be explained in terms of a so-called cage effect, arising from rattling motion in a local molecular cage as a consequence of excluded volume effects. By selectively manipulating the nature of interactions between protein molecules, we determined that excluded volume effects induce sub-diffusive dynamics at sub-microsecond timescales. These findings may help to explain the diffusion-mediated effects of protein crowding on cellular processes.

Differentiating the roles of proteins and polysomes in nucleoid size homeostasis in Escherichia coli

A defining feature of the bacterial cytosolic interior is a distinct membrane-less organelle, the nucleoid, that contains the chromosomal DNA. Although increasing experimental evidence indicates that macromolecular crowding is the dominant mechanism for nucleoid formation, it has remained unclear which crowders control nucleoid volume. It is commonly assumed that polyribosomes play a dominant role, yet the volume fraction of soluble proteins in the cytosol is comparable with that of polyribosomes. Here, we develop a free energy-based model for the cytosolic interior of a bacterial cell to distinguish contributions arising from polyribosomes and cytosolic proteins in nucleoid volume control. The parameters of the model are determined from the existing experimental data. We show that, while the polysomes establish the existence of the nucleoid as a distinct phase, the proteins control the nucleoid volume in physiologically relevant conditions. Our model explains experimental findings in Escherichia coli that the nucleoid compaction curves in osmotic shock measurements do not depend on cell growth rate and that dissociation of polysomes in slow growth rates does not lead to significant nucleoid expansion, while the nucleoid phase disappears in fastest growth rates. Furthermore, the model predicts a cross-over in the exclusion of crowders by their linear dimensions from the nucleoid phase: below the cross-over of 30–50 nm, the concentration of crowders in the nucleoid phase decreases linearly as a function of the crowder diameter, while decreasing exponentially above the cross-over size. Our work points to the possibility that bacterial cells maintain nucleoid size and protein concentration homeostasis via feedback in which protein concentration controls nucleoid dimensions and the nucleoid dimensions control protein synthesis rate.

Determinants for macromolecular crowding-induced thermodynamic stabilization of acid-denatured cytochrome c to molten globules

The macromolecular crowding effect transforms the acid-denatured ferricytochrome c (cyt cIII) (UA-state) to molten-globule (MGMC-state) at pH 1.85. Crowding-induced stabilization free energy (ΔΔG) and preferential hydration ΔΓW were estimated for the UA → MGMC transition. The magnitudes of ΔΔG and ΔΓW were found to be decreased as dextran 70 (D70) > dextran 40 (D40) > ficoll 70 (F70), which demonstrates that ΔΔG and ΔΓW track the molecular size and shape of the crowder towards refolding and stabilization of UA-state to MGMC-state. Analysis of effects of crowders (D40, D70, F70) on thermal and chemical-denaturations of acid-denatured cyt cIII provided several important information, (i) macromolecular crowding increased the thermodynamic stability of acid-denatured cyt cIII, (ii) concentration, size and shape of crowder control the crowding-induced thermodynamic stabilization of MGMC-state, (iii) crowding effect increased the thermal-denaturation midpoint (Tm) with a slight change in enthalpy (ΔHm), suggesting that the steric-excluded volume effect contributes to the crowding-induced increased thermal stability of the acid-denatured protein. Analysis of entropy − enthalpy plots for D40, D70, and F70 reveals that in addition to the steric-excluded volume effect, the enthalpic contribution is also added to the macromolecular crowding-induced stabilization of acid-denatured cyt cIII. The dilute-medium, compound-crowder, purely entropic-crowder and purely enthalpic-crowder curves were obtained for acid-denatured cyt cIII for D70, D40 and F70. The crossover temperature, Tx was calculated from the dilute and compound-crowder curves. The Tx values measured for D40, D70, and F70 were found to be ∼ 250.15 K, 272.15 K, and 275.15 K, respectively, which suggests that the Tx value depends on the size and shape of the crowder. Furthermore, the observation of a lower value of Tx and a minor enthalpic component for D40, D70, and F70 is likely due to the formation of weaker soft interactions of acid-denatured cyt cIII with D40, D70, and F70.

Effects of Homogeneous and Heterogeneous Crowding on Translational Diffusion of Rigid Bovine Serum Albumin and Disordered Alfa-Casein.

Intracellular environment includes proteins, sugars, and nucleic acids interacting in restricted media. In the cytoplasm, the excluded volume effect takes up to 40% of the volume available for occupation by macromolecules. In this work, we tested several approaches modeling crowded solutions for protein diffusion. We experimentally showed how the protein diffusion deviates from conventional Brownian motion in artificial conditions modeling the alteration of medium viscosity and rigid spatial obstacles. The studied tracer proteins were globular bovine serum albumin and intrinsically disordered α-casein. Using the pulsed field gradient NMR, we investigated the translational diffusion of protein probes of different structures in homogeneous (glycerol) and heterogeneous (PEG 300/PEG 6000/PEG 40,000) solutions as a function of crowder concentration. Our results showed fundamentally different effects of homogeneous and heterogeneous crowded environments on protein self-diffusion. In addition, the applied "tracer on lattice" model showed that smaller crowding obstacles (PEG 300 and PEG 6000) create a dense net of restrictions noticeably hindering diffusing protein probes, whereas the large-sized PEG 40,000 creates a "less restricted" environment for the diffusive motion of protein molecules.

Towards the energy landscape of adenylate kinase in crowded milieu: Activity, conformation, structure and dynamics in sequence

Enzyme function is governed by a complex network of conformational changes and internal dynamics, with the same getting more convoluted in the crowded cellular environment. Here, we have explored an intricate interplay amongst activity, structure, conformation, and dynamics of a multidomain enzyme, AK3L1 (UniProtKB: Q9UIJ7) in the crowded milieu. We have monitored changes in the enzyme landscape in response to the chemical denaturant, urea, under the influence of different concentrations of macromolecular crowders. Extensive experimental analyses using FRET-based domain displacement measurements, sub-nanosecond time scale local dynamics, and global structural changes, along with enzymatic activity studies, have been carried out to get deeper insights into the factors that may modulate the functional landscape of adenylate kinase (AK3L1). It was observed that AK3L1 gets activated at low urea concentrations, whereas higher urea concentrations unfold and thereby deactivate the enzyme. A sequential response of AK3L1 is observed towards external perturbation (urea) occurring through a series of well-defined steps. Incorporation of crowders not only shift the maximum activity of enzyme to a higher urea concentration, but also enhance domain compaction, as revealed by FRET studies. The modulation in enzyme activity and solvation dynamics acting as local response, precede global unfolding of the enzyme, indicating that the structural alterations around the active site are quite decoupled from the large amplitude global transitions.

Diffusion NMR and Rheology of a Model Polymer in Bacterial Cell Lysate Crowders.

The intracellular milieu is crowded and heterogeneous, and this can have profound consequences for biomolecule motions and biochemical kinetics. Macromolecular crowding has been traditionally studied in artificial crowders like Ficoll and dextran or globular proteins such as bovine serum albumin. It is, however, not clear if the effects of artificial crowders on such phenomena are the same as the crowding that is experienced in a heterogeneous biological environment. Bacterial cells, for example, are composed of heterogeneous biomolecules with different sizes, shapes, and charges. Using crowders composed of one of three different pretreatments of bacterial cell lysate (unmanipulated, ultracentrifuged, and anion exchanged), we examine the effects of crowding on the diffusivity of a model polymer. We measure the translational diffusivity, via diffusion NMR, of the test polymer polyethylene glycol (PEG) in these bacterial cell lysates. We show that the small (Rg ∼ 5 nm) test polymer shows a modest decrease in self-diffusivity with increasing crowder concentration for all lysate treatments. The corresponding self-diffusivity decrease in the artificial Ficoll crowder is much more pronounced. Moreover, a comparison of the rheological response of biological and artificial crowders shows that while the artificial crowder Ficoll exhibits a Newtonian response even at high concentrations, the bacterial cell lysate is markedly non-Newtonian; it behaves like a shear-thinning fluid with a yield stress. While at any concentration the rheological properties are sensitive to both lysate pretreatment and batch-to-batch variations, the PEG diffusivity is nearly unaffected by the type of lysate pretreatment.

An Impact of Hypoxia and Macromolecular Crowders on Extracellular Matrix Deposition by Human Endometrial Mesenchymal Stromal Cells

The last decades are characterized by intensive development of extracellular matrix (ECM) biology. ECM binds cells in an integral tissue and controls the cell functions – from proliferation and differentiation to migration and apoptosis. Bioactive properties of ECM provide the wide perspectives of using in bioengineering and regenerative medicine. In this context, the ECM production by decellularization of organs, tissues or cell cultures is a key technology. To date, a problem of a rapid and large-scale production of bioactive ECM by cultured cells remains very relevant. Optimization of the ECM deposition conditions by human endometrial mesenchymal stromal cells (MESCs) had not been studied yet. Here, we investigated an impact of macromolecular compounds (crowders) – ficoll and PEG on efficiency of crucial ECM proteins deposition depending on both concentration and molecular weight of crowders under normoxia and hypoxia. According to immunofluorescence analysis, among all studied crowders, ficoll 400 had a potent effect on the production of ECM core proteins – fibronectin, type IV collagen and, in a lower rate, type III collagen. The MESCs incubation under hypoxia promoted the formation of a properly organized ECM structure as well as increase in efficiency of ECM protein deposition. Of note, in these conditions ficoll 400 accelerated the ECM production only in а low serum medium. Together, combination of ficoll 400, low serum medium and hypoxia provides the optimal conditions for ECM synthesis. The present work demonstrates for the first time the phenomenon of macromolecular crowding in the context of improving the conditions for deposition and organization of ECM by MESCs.

Effect of environment and charge patterning on the dynamics of marginally disordered proteins.

Intrinsically disordered proteins (IDPs) and proteins with long intrinsically disordered regions (IDRs) compose a large fraction of the proteome in all domains of life. Despite an extensive body of literature on “ordinary” disordered proteins—ones with high charge to hydropathy ratio—disordered proteins with lower charge to hydropathy ratio remain underinvestigated. To what extent do such “borderline” proteins compactify or perhaps even fold under physiological conditions? In addition, how does the patterning of charged residues in the primary structure of these IDPs affect the three-dimensional structure? Here we aim to characterize three proteins with various degrees of disorder in vitro and in cell using a suite of biophysical techniques. Our results show that the chain dimensions of all three proteins are sensitive to temperature, ionic strength, and crowder concentration in vitro and inside cells, as predicted from analytical and computational models. Moreover, we observe cooperative collapse for two of the three proteins, reminiscent of folding transitions of globular proteins. We further show that charge decoration is the primary predictor of IDP chain dimensions and models that rely solely on the polymer net charge fail to capture the relative chain dimensions of the mutants of the IDPs in our study.

Macromolecular Crowding Is Surprisingly Unable to Deform the Structure of a Model Biomolecular Condensate.

The crowded interior of a living cell makes performing experiments on simpler in vitro systems attractive. Although these reveal interesting phenomena, their biological relevance can be questionable. A topical example is the phase separation of intrinsically disordered proteins into biomolecular condensates, which is proposed to underlie the membrane-less compartmentalization of many cellular functions. How a cell reliably controls biochemical reactions in compartments open to the compositionally-varying cytoplasm is an important question for understanding cellular homeostasis. Computer simulations are often used to study the phase behavior of model biomolecular condensates, but the number of relevant parameters increases as the number of protein components increases. It is unfeasible to exhaustively simulate such models for all parameter combinations, although interesting phenomena are almost certainly hidden in their high-dimensional parameter space. Here, we have studied the phase behavior of a model biomolecular condensate in the presence of a polymeric crowding agent. We used a novel compute framework to execute dozens of simultaneous simulations spanning the protein/crowder concentration space. We then combined the results into a graphical representation for human interpretation, which provided an efficient way to search the model's high-dimensional parameter space. We found that steric repulsion from the crowder drives a near-critical system across the phase boundary, but the molecular arrangement within the resulting biomolecular condensate is rather insensitive to the crowder concentration and molecular weight. We propose that a cell may use the local cytoplasmic concentration to assist the formation of biomolecular condensates, while relying on the dense phase to reliably provide a stable, structured, fluid milieu for cellular biochemistry despite being open to its changing environment.

Size of the protein-protein energy funnel in crowded environment.

Association of proteins to a significant extent is determined by their geometric complementarity. Large-scale recognition factors, which directly relate to the funnel-like intermolecular energy landscape, provide important insights into the basic rules of protein recognition. Previously, we showed that simple energy functions and coarse-grained models reveal major characteristics of the energy landscape. As new computational approaches increasingly address structural modeling of a whole cell at the molecular level, it becomes important to account for the crowded environment inside the cell. The crowded environment drastically changes protein recognition properties, and thus significantly alters the underlying energy landscape. In this study, we addressed the effect of crowding on the protein binding funnel, focusing on the size of the funnel. As crowders occupy the funnel volume, they make it less accessible to the ligands. Thus, the funnel size, which can be defined by ligand occupancy, is generally reduced with the increase of the crowders concentration. This study quantifies this reduction for different concentration of crowders and correlates this dependence with the structural details of the interacting proteins. The results provide a better understanding of the rules of protein association in the crowded environment.

Is Ficoll a Colloid or Polymer? A Multitechnique Study of a Prototypical Excluded-Volume Macromolecular Crowder

The in-cell environment is crowded with macromolecules, and the consequent reduction in free volume, based on the hard-sphere paradigm, is central to understanding macromolecular motions. A much-used model crowder, Ficoll, often assumed to be a compact, if not rigid, colloidal particle, is studied by rheology, small-angle neutron scattering, nuclear magnetic resonance diffusometry, and relaxometry. We find that the Ficoll suspension viscosity scales linearly with concentration cF in the dilute limit and as ∼cF3.8 at high cF, i.e, consistent with the 15/4 (de Gennes) scaling for a reptating polymer. The form factor of Ficoll, obtained via small-angle neutron scattering (SANS), resembles either a Gaussian polymer or a soft polymer blob. From NMR diffusion measurements, we obtain an effective volume fraction for Ficoll that accounts for Ficoll-bound water in two ways and show that each results in a volume occupancy of 60% to 70% in the crowding limit, much larger than the traditionally reported values of around 30%. If we persist with the colloid paradigm and examine the dependence of the zero-q structure factor obtained via SANS in terms of this effective volume fraction, we find that only a combination of particle softness and interparticle attractions, quantified using a computational model, can replicate the experimental S(0). The stark difference between effective and traditional volume occupancies affects the interpretation of previous experiments on macromolecular crowding and might explain the intriguing non-monotonicity observed in the dependence of protein relaxation rates on crowder concentration.

High Macromolecular Crowding in Liposomes from Microfluidics.

The intracellular environment is crowded with macromolecules that influence biochemical equilibria and biomacromolecule diffusion. The incorporation of such crowding in synthetic cells would be needed to mimic the biochemistry of living cells. However, only a few methods provide crowded artificial cells, moreover providing cells with either heterogeneous size and composition or containing a significant oil fraction. Therefore, a method that generates monodisperse liposomes with minimal oil content and tunable macromolecular crowding using polydimethylsiloxane (PDMS)‐based microfluidics is presented. Lipid stabilized water‐in‐oil‐in‐water emulsions that are stable for at least several months and with a high macromolecular crowder concentration that can be controlled with the external osmolality are formed. A crucial feature is that the oil phase can be removed using high flow conditions at any point after production, providing the highly crowded liposomes. Genetically encoded macromolecular crowding sensors show that the high level of macromolecular crowding in the emulsions is fully retained throughout the generation of minimal‐oil lipid bilayers. This modular and robust platform will serve the study of biochemistry under physiologically relevant crowding conditions.

Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles

E. coli cells, which are commonly used to study gene expression, show membraneless organization of gene expression components within the cell. Cell-free expression systems are also widely used to study gene expression, but a major limitation is the lack of a means to spatially organize DNA plasmids and ribosomes to mimic the cellular environment. In this work, we used computer simulations to guide experimental efforts to control the spatial organization of DNA and ribosomes in cell-sized vesicles using macromolecular crowding. Using Brownian dynamics simulations of coarse-grained models of DNA plasmids and crowders, we showed that plasmids remain uniformly distributed at low levels of crowding but become strongly adsorbed at confining surfaces at high levels of crowding. These effects are due to entropic depletion interactions resulting from the presence of crowding molecules. We validated the simulation results experimentally by using fluorescently-labelled DNA plasmids and ribosomes in cell-sized vesicles at different concentrations of the macromolecular crowder Ficoll-70. Large crowder concentrations resulted in preferential localization of plasmids at the walls of the vesicle, while ribosomes remained uniformly distributed throughout the vesicle. We then used kinetic Monte Carlo simulations to study how protein production was affected by crowding-induced changes in spatial organization and diffusion. The localization of DNA to the wall resulted in lower protein abundance and a decrease in translational efficiency with an increase in system size. Experimentally, we tracked the dynamics of transcription and translation in crowded vesicles using a coupled mRNA/protein reporter technique. These results were consistent with results from simulations. We thus used computer simulations to design a cell-free experimental platform capable of spatial organization of gene expression components. This platform can be used to better understand the spatial control of gene expression in cells.

Liquid–Liquid Phase Separation of Peptide/Oligonucleotide Complexes in Crowded Macromolecular Media

The membraneless organelles (MLOs) and coacervates of oppositely charged polyelectrolytes are both formed by liquid-liquid phase separation. To reveal how the crowded cell interior regulates the MLOs, we chose the coacervates formed by peptide S5 and single-stranded oligonucleotide (ss-oligo) at 1:1 charge ratio and investigated the phase separation processes in polyacrylamide (PAM) and poly(ethylene oxide) (PEO) media at varying concentrations. Results show that the droplet formation unit is the neutral primary complex, instead of individual S5 or ss-oligo. Therefore, the coacervation process can be described by the classic theory of nucleation and growth. The dynamic scaling relationships show that S5/ss-oligo coacervation undergoes in sequence the heterogeneous nucleation, diffusion-limited growth, and Brownian motion coalescence with time. The inert crowders generate multiple effects, including accelerating the growth of droplets, weakening the electrostatic attraction, and slowing down or even trapping the droplets in the crowder network. The overall effect is that both the size and size distribution of the droplets decrease with increasing crowder concentration, and the effect of PEO is stronger than that of PAM. Our study provides a further step toward a deeper understanding of the kinetics of MLOs in crowded living cells.

Molecular crowding and conserved interface interactions of human argonaute protein-miRNA-target mRNA complex

RNA interference (RNAi) has been of interest given its role in genetic interference. More significantly, recent studies provided evidence of it being one of the antiviral response mechanisms in humans. Argonaute (Ago) protein plays a central role in the RNA-induced silencing complex (RISC) that cleaves mRNA. Molecular crowding in cellular systems is known to impact dynamics and interactions of biomolecules. We present here the results from our molecular dynamics simulations based study on the interfaces between Ago, miRNA and Target RNA in presence of molecular crowders. 6 simulations at 3 crowder concentrations, including the aqueous condition, were performed. Our results indicate that crowding changes the dynamics, makes the complex stabler and aids binding free energy. More importantly, features conserved across the three systems and amino acid residues with crowding resilient interactions with RNA are identified. Communicated by Ramaswamy H. Sarma

Macromolecular crowding enhances and accelerates extracellular matrix deposition in human umbilical cord derived mesenchymal stem cell cultures

Background & Aim Umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have shown anti-inflammatory, anti-fibrotic and regenerative potential, as well as low immunogenicity. However, direct cell injections, due to low extracellular matrix (ECM) content, are characterized by low cell survival rate and poor localization at the side of implantation. To this end, strategies that enhance and accelerate native ECM synthesis and deposition must be integrated into the developmental cycle of tissue engineering products. Macromolecular crowding (MMC) is a biophysical phenomenon that accelerates thermodynamic activities and biological processes by several orders of magnitude. In cell culture, MMC has been shown to dramatically enhance and accelerate extracellular matrix deposition (e.g. >80-fold within 48 h). Considering that the optimal crowder and crowder concentration is cell-dependent, herein, for first time, we ventured to identify the optimal MMC conditions for hUC-MSCs. Methods, Results & Conclusion hUC-MSCs were seeded at 30,000 cells/cm2 in 24 well plates and allowed to attach for 24 h. Media were then replaced with media containing different carrageenan (κ&λ, κ-LV1, κ-LV2, λ-MV, λ-HV, ι-MV, ι-HV) at various concentrations (0, 1, 5, 10, 50, 100 μg/ml) and 100 μM L-ascorbic acid phosphate. The biophysical properties of the crowders (e.g. dispersity, charge, hydrodynamic radius) were assessed. Cell morphology, viability, metabolic activity and proliferation were analyzed. SDS-PAGE and immunocytochemistry (ICC) analysis for deposited ECM proteins and ELISA for growth factor and immune response were assessed. Biophysical assessment showed for all carrageenan increased particle size with concentration, good polydispersity (at 10-50 μg/ml) and negative charge. ι carrageenan showed poor solubility in all concentrations. No significant difference was observed in cell morphology between the groups. SDS-PAGE and ICC analyses revealed significant increase (p Data to-date indicate the suitability, with respect to enhanced and accelerated ECM deposition, of 10 μg/ml λ carrageenan for hUC-MSCs culture. Therefore, these results provide support for further developing an advanced cell therapy product, which can potentially be used in tissue regeneration for chronic wound healing therapies.

Influence of crowding agents on the dynamics of a multidomain protein in its denatured state: a solvation approach.

It is now well appreciated that the crowded intracellular environment significantly modulates an array of physiological processes including protein folding-unfolding, aggregation, and dynamics to name a few. In this work we have studied the dynamics of domain I of the protein human serum albumin (HSA) in its urea-induced denatured states, in the presence of a series of commonly used macromolecular crowding agents. HSA was labeled at Cys-34 (a free cysteine) in domain I with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN) to act as a solvation probe. In partially denatured states (2-6M urea), lower crowder concentrations (~ < 125g/L) induced faster dynamics, while the dynamics became slower beyond 150g/L of crowders. We propose that this apparent switch in dynamics is an evidence of a crossover from soft (enthalpic) to hard-core (entropic) interactions between the protein and crowder molecules. That soft interactions are also important for the crowders used here was further confirmed by the appreciable shift in the wavelength of the emission maximum of BADAN, in particular for PEG8000 and Ficoll 70 at concentrations where the excluded volume effect is not dominant.

Crowding-Induced DNA Translocation through a Protein Nanopore.

A crowded cellular environment is highly associated with many significant biological processes. However, the effect of molecular crowding on the translocation behavior of DNA through a pore has not been explored. Here, we use nanopore single-molecule analytical technique to quantify the thermodynamics and kinetics of DNA transport under heterogeneous cosolute PEGs. The results demonstrate that the frequency of the translocation event exhibits a nonmonotonic dependence on the crowding agent size, while both the event frequency and translocation time increase monotonically with increasing crowder concentration. In the presence of PEGs, the rate of DNA capture into the nanopore elevates 118.27-fold, and at the same time the translocation velocity decreases from 20 to 120 μs/base. Interestingly, the impact of PEG 4k on the DNA-nanopore interaction is the most notable, with up to ΔΔG = 16.27 kJ mol-1 change in free energy and 764.50-fold increase in the binding constant at concentration of 40% (w/v). The molecular crowding effect will has broad applications in nanopore biosensing and nanopore DNA sequencing in which the strategy to capture analyte and to control the transport is urgently required.

The effects of polydisperse crowders on the compaction of the Escherichia coli nucleoid.

DNA binding proteins, supercoiling, macromolecular crowders, and transient DNA attachments to the cell membrane have all been implicated in the organization of the bacterial chromosome. However, it is unclear what role these factors play in compacting the bacterial DNA into a distinct organelle-like entity, the nucleoid. By analyzing the effects of osmotic shock and mechanical squeezing on Escherichia coli, we show that macromolecular crowders play a dominant role in the compaction of the DNA into the nucleoid. We find that a 30% increase in the crowder concentration from physiological levels leads to a three-fold decrease in the nucleoid's volume. The compaction is anisotropic, being higher along the long axes of the cell at low crowding levels. At higher crowding levels, the nucleoid becomes spherical, and its compressibility decreases significantly. Furthermore, we find that the compressibility of the nucleoid is not significantly affected by cell growth rates and by prior treatment with rifampicin. The latter results point out that in addition to poly ribosomes, soluble cytoplasmic proteins have a significant contribution in determining the size of the nucleoid. The contribution of poly ribosomes dominates at faster and soluble proteins at slower growth rates.