Effects of Macromolecular Crowding on the Collapse of Biopolymers
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Experiments show that macromolecular crowding modestly reduces the size of intrinsically disordered proteins (IDPs) even at volume fraction ($\phi$) similar to that in the cytosol whereas DNA undergoes a coil-to-globule transition at very small $\phi$. We show using a combination of scaling arguments and simulations that the polymer size $\overline{R}_g(\phi)$ depends on $x = \overline{R}_g(0)/D$ where $D$ is the $\phi$-dependent distance between the crowders. If $x\lesssim \mathcal{O}(1)$, there is only a small decrease in $\overline{R}_g(\phi)$ as $\phi$ increases. When $x\gg \mathcal{O}(1)$, a cooperative coil-to-globule transition is induced. Our theory quantitatively explains a number of experiments.Keywords:
Macromolecular Crowding
Intrinsically Disordered Proteins
Scaling law
Volume fraction
The non-ideal properties of solutions containing high concentrations of macromolecules can result in enormous increases in the activity of the individual macromolecules. There is considerable evidence that macromolecular crowding and confinement not only occur in cells, but that these are major determinants of the activity of the proteins and other intracellular macromolecules. This concept has important implications for cell volume regulation because, under crowded conditions, relatively small changes in concentration, consequent to alterations of water content, lead to large changes in macromolecular activity which could provide a mechanism by which cells sense changes in their volume. This brief review considers 1) direct demonstrations that introducing a high concentration of appropriate macromolecules into cells in vitro produced volume regulatory changes, 2) the physical chemical principles involved in the effects of crowding of macromolecules on their activity, 3) estimates of the actual intracellular activity of macromolecules, 4) a proposed model of how changes in macromolecular crowding could signal volume regulation in cells, and 5) brief consideration of the complexities introduced by interactions between macromolecules, water and cosolutes.The hypothesis that macromolecular crowding provides a mechanism by which cells sense changes in their volume is plausible and is supported by striking observations in red blood cell ghosts and perfused barnacle muscle cells. However, the signaling molecules involved have not been identified, the proposed model is not fully consistent with the experiments, experimental verification in intact cells is lacking, and numerous alternative or additional mechanisms are not excluded.
Macromolecular Crowding
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Significance Cells contain 30–40% dissolved protein and RNA by volume. In vivo protein binding and stability might differ significantly from in vitro measurements. Crowding by the excluded volume of these macromolecules has been studied extensively by experiment and theory. When the crowding effects of macromolecules, water, and ions are treated on an equal footing, the effect is opposite to that commonly believed. Large molecules are less effective at crowding than water and ions. There is also a surprisingly weak dependence on crowder size. Molecules of medium size have the same effect as much larger macromolecules like proteins and RNA. These results require a reassessment of observed high-concentration effects and of strategies to mimic in vivo conditions with in vitro experiments.
Macromolecular Crowding
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Macromolecular Crowding
Intrinsically Disordered Proteins
Folding (DSP implementation)
Crowding
Macromolecular Substances
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Replisome
Macromolecular Crowding
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Macromolecular crowding and confinement, the effects caused by high concentrations
of macromolecules in solution and/or in small compartments, are believed to influence
diffusion processes, intermolecular interactions, protein folding, and intracellular
transport in living cells. Understanding mechanisms of transport in biological systems
(such as living cells) is complex and challenging. We construct cell mimetic environments
in which the artificial macromolecules (e.g. polyethylene glycol, Ficoll70) are
compartmentalized not in cells but in concentrated environments and agarose gel
networks. In this work we have established a system to generate stable and monodisperse
droplets of hierarchical confinement. The goal of this study is to measure
translational diffusion in crowded and confined geometries of varying concentrations
of different macromolecules on diffusion. We have combined the use of pulsed-fieldgradient
nuclear magnetic resonance (PFG NMR) with small-angle neutron scattering
(SANS) in order to obtain new insights in simple model systems of macromolecular
crowding. The NMR and SANS techniques complement each other. Using PFG NMR
technique, we have monitored the dynamics of synthetic macromolecules with multiple
chemical components in complex environments. SANS, on the other hand, yields
structure (size) of macromolecules. Our experimental findings in cell mimetic environments
provide an important step towards gaining further insights into the effects
of macromolecular crowding on diffusion and conformation.
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Abstract Most biologically relevant environments involve highly concentrated macromolecular solutions and most biological processes involve macromolecules that diffuse and interact with other macromolecules. Macromolecular crowding is a general phenomenon that strongly affects the transport properties of macromolecules (rotational and translational diffusion) as well as the position of their equilibria. NMR methods can provide information on molecular interactions, as well as on translational and rotational diffusion. In fact, rotational diffusion, through its determinant role in NMR relaxation, places a practical limit on the systems that can be studied by NMR. While in dilute solutions of non‐aggregating macromolecules this limit is set by macromolecular size, in crowded solutions excluded volume effects can have a strong effect on the observed diffusion rates. Hydrodynamic theory offers some insight into the magnitude of crowding effects on NMR observable parameters. Copyright © 2004 John Wiley & Sons, Ltd.
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Macromolecular Crowding
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Macromolecular Crowding
Percolation (cognitive psychology)
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Inside cells, the concentration of macromolecules can reach up to 400 g/L. In such crowded environments, proteins are expected to behave differently than in vitro. It has been shown that the stability and the folding rate of a globular protein can be altered by the excluded volume effect produced by a high density of macromolecules. However, macromolecular crowding effects on intrinsically disordered proteins (IDPs) are less explored. These proteins can be extremely dynamic and potentially sample a wide ensemble of conformations under non-denaturing conditions. The dynamic properties of IDPs are intimately related to the timescale of conformational exchange within the ensemble, which govern target recognition and how these proteins function. In this work, we investigated the macromolecular crowding effects on the dynamics of several IDPs by measuring the NMR spin relaxation parameters of three disordered proteins (ProTα, TC1, and α-synuclein) with different extents of residual structures. To aid the interpretation of experimental results, we also performed an MD simulation of ProTα. Based on the MD analysis, a simple model to correlate the observed changes in relaxation rates to the alteration in protein motions under crowding conditions was proposed. Our results show that 1) IDPs remain at least partially disordered despite the presence of high concentration of other macromolecules, 2) the crowded environment has differential effects on the conformational propensity of distinct regions of an IDP, which may lead to selective stabilization of certain target-binding motifs, and 3) the segmental motions of IDPs on the nanosecond timescale are retained under crowded conditions. These findings strongly suggest that IDPs function as dynamic structural ensembles in cellular environments.
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