High Concentrations Of Macromolecules Research Articles

Effect of macromolecules on the viscosity of whole condensed milk

The viscosity of a fluid refers to the resistance to flow, such as in the case of whole condensed milk. Condensed milk is a product created by partially dehydrating milk, concentrated milk, or reconstituted milk, and then sugar is added. Its fat and protein content can be adjusted to meet specific characteristics. The aim of this study was to evaluate the effect of macromolecules on the viscosity of various brands of whole condensed milk. The brands of whole condensed milk analyzed were classified as LCA, LCB, LCC, LCD, and LCE, with a total of 50 samples from different batches. The viscosity was measured using the Ford cup method, which was validated through tests for linearity, detection limit, quantification limit, and repeatability. The physicochemical tests included viscosity, soluble solids (oBrix), moisture, protein, lipids, RMF, carbohydrates, total caloric value, and SNG. The main findings showed that the LCC, LCD, and LCE brands had high levels of macromolecules (protein, lipids, and carbohydrates) and viscosity. Consequently, it was concluded that high concentrations of macromolecules in whole condensed milk result in increased viscosity of the product.

A Difference between In Vitro and In-Cell Protein Dimer Formation.

The high concentration of macromolecules in cells affects the stability of proteins and protein complexes via hard repulsions and chemical interactions, yet few studies have focused on chemical interactions. We characterized the domain-swapped dimer of the B1 domain of protein G in buffer and Escherichia coli cells by using heteronuclear, multidimensional nuclear magnetic resonance spectroscopy. In buffer, the monomer is a partially folded molten globule, but that species is not observed in cells. Experiments using urea suggest that the monomer is unfolded in cells, but again, the molten-globule form of the monomer is absent. The data suggest that attractive chemical interactions in the cytoplasm unfold the molten globule. We conclude that the intracellular environment not only modulates the stability of protein complexes but also can change the species present, reinforcing the idea that chemical interactions are more important than hard repulsions in cells.

Molecular Crowding Modulates Actin Filament Mechanics and Structure

The cellular environment is crowded with high concentrations of macromolecules that significantly reduce the accessible volume limiting biomolecule interactions. Reductions in cellular volume can generate depletion forces that affect protein assembly and stability. The mechanical and structural properties of actin filaments play critical roles in various cellular functions including structural support, cell movement, division, and intracellular transport. Although the effects of molecular crowding on actin polymerization have been shown, how crowded environments affect filament conformations, dynamics, and mechanical properties is unknown. In this study, we investigate the effects of solution crowding on the modulations of filament mechanics and structure both in vitro and in silico. Direct visualization of filaments in the presence of crowding agents is achieved by fluorescence microscopy imaging, allowing for the quantification of filament thermal bending dynamics and mechanics. Biophysical analysis indicate that molecular crowding modulates thermal fluctuations, enhances filament's effective bending stiffness, and reduces average filament lengths. Utilizing the explicit molecular dynamics simulations, we demonstrate that molecular crowding alters filament conformations and structural properties indicating overall compaction and stabilization that are directly coupled to filament mechanics. Taken together, our study suggests the interplay between excluded volume effects and non‐specific interactions raised from molecular crowding may modulate actin filament mechanics and structure.Support or Funding InformationThis study was supported by the UCF start‐up fund and In‐House grant for H.K. The authors would like to thank the National Science Foundation for support of this work through REU site EEC 1560007. We acknowledge the computational time from UCF stokes cluster.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Actin Filament Mechanics and Structure in Crowded Environments.

The cellular environment is crowded with high concentrations of macromolecules that significantly reduce accessible volume for biomolecular interactions. Reductions in cellular volume can generate depletion forces that affect protein assembly and stability. The mechanical and structural properties of actin filaments play critical roles in various cellular functions, including structural support, cell movement, division, and intracellular transport. Although the effects of molecular crowding on actin polymerization have been shown, how crowded environments affect filament mechanics and structure is unknown. In this study, we investigate the effects of solution crowding on the modulations of actin filament bending stiffness and conformations both in vitro and in silico. Direct visualization of thermally fluctuating filaments in the presence of crowding agents is achieved by fluorescence microscopy imaging. Biophysical analysis indicates that molecular crowding enhances filament's effective bending stiffness and reduces average filament lengths. Utilizing the all-atom molecular dynamics simulations, we demonstrate that molecular crowding alters filament conformations and intersubunit contacts that are directly coupled to the mechanical properties of filaments. Taken together, our study suggests that the interplay between excluded volume effects and nonspecific interactions raised from molecular crowding may modulate actin filament mechanics and structure.

Role of Conformational Flexibility in Monte Carlo Simulations of Many-Protein Systems.

Efficient computational modeling of biological systems characterized by high concentrations of macromolecules often relies on rigid-body Brownian Dynamics or Monte Carlo (MC) simulations. However, the accuracy of rigid-body models is limited by the fixed conformation of the simulated biomolecules. Multi-conformation Monte Carlo (mcMC) simulations of protein solutions incorporate conformational flexibility via a conformational swap trial move within a predetermined library of discrete protein structures, thereby alleviating artifacts arising from the use of a single protein conformation. Here, we investigate the impact of the number of distinct protein structures in the conformational library and the extent of conformational sampling used in its generation on structural observables computed from simulations of hen egg white lysozyme (HEWL), human γD-Crystallin, and bovine γB-Crystallin solutions. We find that the importance of specific protocols for the construction of the protein structure library is strongly dependent on the nature of the simulated system.

Facilitated Diffusion Mechanisms in DNA Base Excision Repair and Transcriptional Activation.

Preservation of the coding potential of the genome and highly regulated gene expression over the life span of a human are two fundamental requirements of life. These processes require the action of repair enzymes or transcription factors that efficiently recognize specific sites of DNA damage or transcriptional regulation within a restricted time frame of the cell cycle or metabolism. A failure of these systems to act results in accumulated mutations, metabolic dysfunction, and disease. Despite the multifactorial complexity of cellular DNA repair and transcriptional regulation, both processes share a fundamental physical requirement that the proteins must rapidly diffuse to their specific DNA-binding sites that are embedded within the context of a vastly greater number of nonspecific DNA-binding sites. Superimposed on the needle-in-the-haystack problem is the complex nature of the cellular environment, which contains such high concentrations of macromolecules that the time frame for diffusion is expected to be severely extended as compared to dilute solution. Here we critically review the mechanisms for how these proteins solve the needle-in-the-haystack problem and how the effects of cellular macromolecular crowding can enhance facilitated diffusion processes. We restrict the review to human proteins that use stochastic, thermally driven site-recognition mechanisms, and we specifically exclude systems involving energy cofactors or circular DNA clamps. Our scope includes ensemble and single-molecule studies of the past decade or so, with an emphasis on connecting experimental observations to biological function.

Crowding, Entropic Forces, and Confinement: Crucial Factors for Structures and Functions in the Cell Nucleus.

The view of the cell nucleus as a crowded system of colloid particles and that chromosomes are giant self-avoiding polymers is stimulating rapid advances in our understanding of its structure and activities, thanks to concepts and experimental methods from colloid, polymer, soft matter, and nano sciences and to increased computational power for simulating macromolecules and polymers. This review summarizes current understanding of some characteristics of the molecular environment in the nucleus, of how intranuclear compartments are formed, and of how the genome is highly but precisely compacted, and underlines the crucial, subtle, and sometimes unintuitive effects on structures and reactions of entropic forces caused by the high concentration of macromolecules in the nucleus.

Brownian Dynamics Computational Model of Protein Diffusion in Crowded Media with Dextran Macromolecules as Obstacles

The high concentration of macromolecules (i.e., macromolecular crowding) in cellular environments leads to large quantitative effects on the dynamic and equilibrium biological properties. These effects have been experimentally studied using inert macromolecules to mimic a realistic cellular medium. In this paper, two different experimental in vitro systems of diffusing proteins which use dextran macromolecules as obstacles are computationally analyzed. A new model for dextran macromolecules based on effective radii accounting for macromolecular compression induced by crowding is proposed. The obtained results for the diffusion coefficient and the anomalous diffusion exponent exhibit good qualitative and generally good quantitative agreement with experiments. Volume fraction and hydrodynamic interactions are found to be crucial to describe the diffusion coefficient decrease in crowded media. However, no significant influence of the hydrodynamic interactions in the anomalous diffusion exponent is found.

Macromolecular and Small Molecular Crowding Have Similar Effects on α-Synuclein Structure.

The intracellular milieu contains upwards of 400 g of macromolecules per liter. This crowding is thought to have a larger influence on intrinsically disordered proteins, whose chains are expanded, than on compact globular proteins. Classic theories of macromolecular crowding predict that increasing excluded volume effects will lead disordered proteins to compaction, and a great deal of data, from both simulation and experiments support this idea. We used nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopies to characterize the structure and fibrillation of α-synuclein, an intrinsically disordered protein implicated in Parkinson's disease, using Ficoll70, its monomer sucrose and bovine serum albumin as crowding agents. Surprisingly, volume exclusion induced by high concentrations of macromolecules may not be the main reason for the compaction of α-synuclein. Our results indicate that all aspects crowding must be considered to understand protein conformation under crowded conditions.

Implications of Volume Exclusion: A Look at Thermodynamic Perspective of DNA-Hemoglobin Complexes and Their Reconstitutes Under Macromolecular Crowding.

Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo. Another significant factor which is overlooked is the effects of macromolecular crowding and its consequences in the actual biochemical and physiological environment. Such influences of crowding, its modification and physiological parameters have been reported. The present study investigates the effect of molecular crowding on binding characteristics of Salmon sperm DNA with Bovine hemoglobin and their reconstitutes in presence of molecular crowders viz., Poly ethylene glycol (PEG) and Dextran of different molecular weight by fluorescence, UV visible spectroscopic technique at different temperatures. The results showed that BHb fluorescence was quenched by sDNA through static quenching mechanism which is enhanced in presence of polymers. The number of binding sites 'n' and binding constants 'K' were determined at different temperatures based on fluorescence quenching. The thermodynamic parameters namely ∆H°, ∆G°, T∆S° were studied at different temperatures and the results indicate that hydrophobic forces are predominant in the sDNA-BHb complex. Negative ∆G° values imply that the binding process is spontaneous.

Relative Cosolute Size Influences the Kinetics of Protein-Protein Interactions

Protein signaling occurs in crowded intracellular environments, and while high concentrations of macromolecules are postulated to modulate protein-protein interactions, analysis of their impact at each step of the reaction pathway has not been systematically addressed. Potential cosolute-induced alterations in target association are particularly important for a signaling molecule like calmodulin (CaM), where competition among >300 targets governs which pathways are selectively activated. To explore how high concentrations of cosolutes influence CaM-target affinity and kinetics, we methodically investigated each step of the CaM-target binding mechanism under crowded or osmolyte-rich environments mimicked by ficoll-70, dextran-10, and sucrose. All cosolutes stabilized compact conformers of CaM and modulated association kinetics by affecting diffusion and rates of conformational change; however, the results showed that differently sized molecules had variable effects to enhance or impede unique steps of the association pathway. On- and off-rates were modulated by all cosolutes in a compensatory fashion, producing little change in steady-state affinity. From this work insights were gained on how high concentrations of inert crowding agents and osmolytes fit into a kinetic framework to describe protein-protein interactions relevant for cellular signaling.

Enthalpy Mediated Protein Stabilization by Macromolecular Crowding

Understanding the structure and stability of biomolecules in the cellular environment requires knowledge of their interplay with high concentrations of macromolecules, osmolytes, salts and membranes. The macromolecular crowding effect in a cell is usually described by the excluded volume theory based on hardcore repulsions [1]. However, recent studies emphasize the role of further contributions aside from a pure volume effect including enthalpic and solvent effects [2, 3]. We study cosolute effects at high molecular and macromolecular concentrations via a thermodynamic analysis of the thermal unfolding of ubiquitin in the presence of different concentrations of cosolutes (glucose, dextran, poly(ethylene glycol), potassium chloride) [4]. In contrast to the excluded volume theory, we observed enthalpic stabilization and entropic destabilization forces for all tested cosolutes. The enthalpic stabilization mechanism of ubiquitin in macromolecular polysaccharide solutions of dextran was thereby similar to the effects observed in monomeric glucose. The more hydrophobic polymer polyethylene glycol destabilizes native ubiquitin, presumably via unspecific direct interactions with the protein. Thereby, the macromolecular character of PEG and dextran - and hence its excluded volume - is not to the determining factor which influences the folding equilibrium of ubiquitin. Instead, our data suggest a key role for hydration water molecules in the interaction of proteins and cosolutes.

Tuning of Structure-Function Relationships by Macromolecular Crowding

The cellular interior is crowded with high concentrations of macromolecules and small organic solutes and strict maintenance of this environment, where ∼30% of the total volume is occupied, is essential to cellular function. The volume occupied by these molecules confines the 3D space in which any particular protein can reside and is thought to modulate protein-protein interactions by affecting diffusional encounter and reaction rates. In this work, we investigate the mechanistic consequences of crowded environments on the structure-function relationship of the ubiquitous protein calmodulin (CaM). CaM is highly flexible and dynamic, making it particularly susceptible to crowded environments, and its conformational plasticity is essential for accommodating binding to its 300 identified targets. We determined that crowded environments, created with purified polymer systems and sucrose, stabilize compact conformers of CaM, reduce translational and rotational diffusion, modulate association and dissociation kinetics with a calmodulin-binding target, and control rates of conformational transition. Most notably, these effects are determined by the size and concentration of the crowding reagent indicating that the composition of the environment differentially tunes CaM structure, dynamics, and target association kinetics. Our results are shown in a general kinetic framework where there are consequences for target selectivity and cellular signaling.

Are current atomistic force fields accurate enough to study proteins in crowded environments?

The high concentration of macromolecules in the crowded cellular interior influences different thermodynamic and kinetic properties of proteins, including their structural stabilities, intermolecular binding affinities and enzymatic rates. Moreover, various structural biology methods, such as NMR or different spectroscopies, typically involve samples with relatively high protein concentration. Due to large sampling requirements, however, the accuracy of classical molecular dynamics (MD) simulations in capturing protein behavior at high concentration still remains largely untested. Here, we use explicit-solvent MD simulations and a total of 6.4 µs of simulated time to study wild-type (folded) and oxidatively damaged (unfolded) forms of villin headpiece at 6 mM and 9.2 mM protein concentration. We first perform an exhaustive set of simulations with multiple protein molecules in the simulation box using GROMOS 45a3 and 54a7 force fields together with different types of electrostatics treatment and solution ionic strengths. Surprisingly, the two villin headpiece variants exhibit similar aggregation behavior, despite the fact that their estimated aggregation propensities markedly differ. Importantly, regardless of the simulation protocol applied, wild-type villin headpiece consistently aggregates even under conditions at which it is experimentally known to be soluble. We demonstrate that aggregation is accompanied by a large decrease in the total potential energy, with not only hydrophobic, but also polar residues and backbone contributing substantially. The same effect is directly observed for two other major atomistic force fields (AMBER99SB-ILDN and CHARMM22-CMAP) as well as indirectly shown for additional two (AMBER94, OPLS-AAL), and is possibly due to a general overestimation of the potential energy of protein-protein interactions at the expense of water-water and water-protein interactions. Overall, our results suggest that current MD force fields may distort the picture of protein behavior in biologically relevant crowded environments.

Time-Dependent Extracellular Matrix Organization and Secretion from Vascular Endothelial Cells due to Macromolecular Crowding

ABSTRACTInteractions between biological cells and surrounding extracellular matrix (ECM) materials modulate many cell behaviors including adhesion and migration. One key example of this cellmatrix reciprocity is in the context of angiogenesis, the sprouting of new blood vessels from preexisting vasculature. Vascular endothelial cells (VECs) create and remodel the ECM during this process. In vivo, the surrounding fluid environment includes high concentrations of macromolecules, and is considered “crowded” in comparison to in vitro environments. Here, we quantified the amount and organization of collagen IV, a prominent ECM component of VECs, that was produced by these cells over four weeks in vitro in the presence or absence of macromolecular crowder (MMC) nanoparticles that approximated in vivo crowding. In the presence of MMCs, the amount and degree of alignment of collagen IV was greater. This ECM difference emerged within one week and was sustained for over four weeks. We explored the effect of initial cell density (cells/µm2) on this matrix production, to consider potential differences at a wound site versus an intact vessel. Moreover, we found the biophysical effect of MMCs to be unmodulated by secretions from an adjacent cell type in microvessels (pericytes). These results suggest that macromolecular crowding plays a direct role in remodeling the basement membrane, and that such crowding can be induced in vitro to more closely approximate the cell microenvironment.

Treatment of a Malaysian Leachate Sample Using Electrocoagulation

A real leachate sample, taken from a Kuala Lumpur suburban area, was used in order to investigate the efficiency of electrocoagulation using Fe electrodes as both cathodes and anodes to reduce chemical oxygen demand (COD) and color of the sample. The effects of pH value and current density on the efficiency of the leachate treatment were investigated. Meanwhile, it was well understood that using this approach for the treatment of the studied leachate could successfully lower the COD value and color of the leachate wastewater by 81% and 72%, respectively. To the knowledge of the authors, to date, no real leachate samples have been treated by electrocoagulation methods elsewhere. Reference (2) has extensively covered the advantages and drawbacks of various contemporary approaches in landfill leachate treatments. Compared to current treatment processes available today, electrocoagulation can benefit from equipment simplicity; potable, clear, colorless and odorless treated product (6); much larger and stable flocs containing less bound water showing sufficient acid resistance compared to chemical flocs which enables them to be filtered by means of membranes; production of effluents with less total dissolved solids (TDS) content compared to chemical treatments which results in lower water recovery cost in case of water reuse (8); the capability of removing the smallest colloidal particles due to the facilitated coagulation by means of applied electric field (5 ); the absence of chemicals and as a result no secondary pollution which is common in membrane cleaning processes and many other treatment systems. Reference (6) investigated the use of electrocoagulation to treat landfill leachate using surface response methodology. They studied the effects of current density, pH, time and fluid conductivity and the interaction between them to get an optimal turbidity removal. Reference (7) used aluminium plates to electrocoagulate a leachate which had showed low biodegradability and high concentration of macromolecules and then made a quantitative comparison with classical coagulation-flocculation with aluminium ions and also electrolysis alone. This article investigates the application of electrocoagulation in the treatment of a real sample of landfill leachate. The experiments in this study were all carried out in a batch mode in order to evaluate the effects of different operational variables including leachate pH and current density and their effects on the COD and color removal of the leachate wastewater.

Protein Disorder Prevails under Crowded Conditions

Crowding caused by the high concentrations of macromolecules in the living cell changes chemical equilibria, thus promoting aggregation and folding reactions of proteins. The possible magnitude of this effect is particularly important with respect to the physiological structure of intrinsically disordered proteins (IDPs), which are devoid of well-defined three-dimensional structures in vitro. To probe this effect, we have studied the structural state of three IDPs, α-casein, MAP2c, and p21(Cip1), in the presence of the crowding agents Dextran and Ficoll 70 at concentrations up to 40%, and also the small-molecule osmolyte, trimethylamine N-oxide (TMAO), at concentrations up to 3.6 M. The structures of IDPs under highly diluted and crowded conditions were compared by a variety of techniques, fluorescence spectroscopy, acrylamide quenching, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, fluorescence correlation spectroscopy (FCS), and far-UV and near-UV circular dichroism (CD) spectroscopy, which allow us to visualize various levels of structural organization within these proteins. We observed that crowding causes limited structural changes, which seem to reflect the functional requirements of these IDPs. α-Casein, a protein of nutrient function in milk, changes least under crowded conditions. On the other hand, MAP2c and, to a lesser degree, p21(Cip1), which carry out their functions by partner binding and accompanying partially induced folding, show signs of local structuring and also some global compaction upon crowded conditions, in particular in the presence of TMAO. The observations are compatible with the possible preformation of binding-competent conformations in these proteins. The magnitude of these changes, however, is far from that of the cooperative folding transitions elicited by crowding in denatured globular proteins; i.e., these IDPs do remain in a state of rapidly interconverting structural ensemble. Altogether, our results underline that structural disorder is the physiological state of these proteins.

Effect of Crowding by Dextrans on the Hydrolysis of N-Succinyl-l-phenyl-Ala-p-nitroanilide Catalyzed by α-Chymotrypsin

Traditionally, studies on the diffusion-controlled reaction of biological macromolecules have been carried out in dilute solutions (in vitro). However, in an intracellular environment (in vivo), there is a high concentration of macromolecules, which results in nonspecific interactions (macromolecular crowding). This affects the kinetics and thermodynamics of the reactions that occur in these systems. In this paper, we study the crowding effect of large macromolecules on the reaction rates of the hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by α-chymotrypsin, by adding dextrans of various molecular weights to the reaction solutions. The results indicate that the volume occupied by the crowding agent, but not its size, plays an important role in the rate of this reaction. A v(max) decay and a K(m) increase were obtained when the dextran concentration in the sample was increased. The increase in K(m) can be attributed to the slowing of protein diffusion, due to the presence of crowding. Whereas the decrease in v(max) could be explained by the effect of mixed inhibition by product, which is enhanced in crowded media. As far as we know, this is the first reported experiment on the crowding effect in an enzymatic reaction with a mixed inhibition by product.

Study of organic matter during coagulation and electrocoagulation processes: Application to a stabilized landfill leachate

Organic matter contained in leachates appears to be a relevant indicator of waste evolution. Among the physico-chemical treatments applied to stabilize leachates, coagulation–flocculation is considered a classical process and electrocoagulation can be developed. Electrocoagulation tests were carried out in a laboratory pilot using aluminium plates and compared to classical coagulation–floculation with aluminium ions and to electrolysis alone. The leachate used in this study came from the landfill of Crezin (Limoges, France) and it presented low biodegradability and high concentration of macromolecules. To assess the efficiency of the chemical and electrochemical processes, we specifically studied Organic Matter (OM) by using two protocols: adsorption on XAD resins and fractionation by ultrafiltration. Biodegradable Dissolved Organic Carbon (BDOC) measurements were applied to the fractions extracted by XAD resins and were also used as an indicator of the efficiency of the treatments. Residual organic matter concentration was the same for the two processes but its composition appeared different after electrocoagulation: a higher percentage of small hydrophilic organic molecules which seemed to be less biodegradable than the initial organic matter was observed.

Isolation of cell nuclei using inert macromolecules to mimic the crowded cytoplasm.

Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v) or 70 kDa dextran (35% w/v) to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 µM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II) were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox that the small-scale motion and irregular conformation of chromatin seen in vivo are not reproduced in nuclei isolated in conventional ionic media.