Is the depletion force entropic? Molecular crowding beyond steric interactions

Sapir, Liel; Harries, Daniel

doi:10.1016/j.cocis.2014.12.003

Cited by 119 publications

(137 citation statements)

References 79 publications

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“…Clearly, synthetic polymers are poor mimics of the cellular interior, and existing theories need to be modified. The modifications must account for nonspecific chemical attractions that act enthalpically to destabilize the protein and nonspecific repulsive chemical interactions that act enthalpically to stabilize the protein (22). The picture is even more complicated because the accounting must also consider solvent (23), including its size relative to the crowding molecules (21).…”

Section: Synthetic Polymer and Their Monomers Are Not Biologically Rementioning

confidence: 99%

In-cell thermodynamics and a new role for protein surfaces

Smith

Zhou

Gorensek

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

147

223

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There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.protein NMR | protein thermodynamics | protein folding | in-cell NMR C lassic theories about the effects of complex environments consider only hard-core repulsions (volume exclusion) and so predict entropy-driven protein stabilization (1-3). Here, we use the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) as a model to test this idea in living cells. SH3 exists in a dynamic equilibrium between the folded state and the unfolded ensemble (4). This two-state behavior (5) is ideal for NMR-based studies of folding. Fluorine labeling (6) of its sole tryptophan leads to only two 19 F resonances (7): one from the folded state, the other from the unfolded ensemble (Fig. 1A). The area under each resonance is proportional to its population, ρ f and ρ u, respectively. These populations are used to quantify protein stability via the modified standard state free energy of unfolding,where R is the gas constant and T is the absolute temperature. Furthermore, the width at half height of each resonance is proportional to the transverse relaxation rate, which is an approximate measure of intermolecular interactions (8-10). Thus, this simple system yields both quantitative thermodynamic knowledge and information about interactions involving the folded state and the unfolded ensemble.To assess the enthalpic (ΔH°′ U ) and entropic (ΔS°′ U ) components, we measured the temperature dependence of ΔG°′ U .

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Section: Synthetic Polymer and Their Monomers Are Not Biologically Rementioning

confidence: 99%

In-cell thermodynamics and a new role for protein surfaces

Smith

Zhou

Gorensek

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

147

223

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“…1 A and B predicts a generic "baseline" stabilization of all folded protein states, all higher oligomer states, and all ligand-protein bound states, whereas the evidence suggests very variable degrees of effect. Second, size effects should be purely entropic, whereas enthalpic contributions are often seen (6,7). Third, crucially, in an earlier study Pielak and coworkers (6) did not find the dependence on crowding molecule size predicted by the conventional model of Fig.…”

mentioning

confidence: 99%

Unpacking the origins of in-cell crowding

Sharp

2016

Proc. Natl. Acad. Sci. U.S.A.

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The living cell, the fundamental unit of biological organization, was discovered in its apparent simplicity by van Leeuwenhoek in the 17th century. Scientists since then have continued to unpack nested levels of amazingly complex cellular structure and organization, right down to the atomic level. However, one of the cell's simplest properties is not yet understood, perhaps even misunderstood. The interior of the cell contains a high concentration of dissolved solutes, principally ions, metabolites, proteins, and RNA. In a word, it is crowded in there. Estimates range from 30% to 40% by volume occupied by protein and RNA solutes, depending on the cell type and compartment (1). Biochemists and biophysicists have long been concerned that these high concentrations impart significantly nonideal solution behavior. The vast majority of measurements of equilibria and rates have been made in vitro. Although the ionic strength, osmolality, pH, and redox potential environment inside the cell can be matched with suitable buffers, the concern is that the effects of the macromolecular solutes, principally protein and RNA, are not accounted for. The crucial and currently unanswered questions are, How relevant is the plethora of in vitro experiments to in vivo conditions? Are measured equilibrium constants, rates, and other thermodynamic data significantly different? If so, by how much and in what direction? In principle one can address this directly by in vivo measurements, but the experiments are difficult, and the answers have been slow in coming. In PNAS Smith et al. (2), by measuring a protein folding equilibrium in vivo, now provide some answers-and they are surprising.To appreciate the surprise, it is helpful to briefly touch on the more than three-decade history of the macromolecular crowding field. The notable feature of macromolecules as cosolvents that is missing from simple buffers is their size. All molecules in solution effectively exclude each other; they cannot overlap, due to what is termed the hard-core or van der Waals repulsion. An early pioneer in this field, Minton (3), explicitly emphasized "excluded volume as a determinant of macromolecular structure and reactivity," whereas Ellis (4) stressed the "obvious" aspect of this, meaning that a large excluded volume is a property of all macromolecules. An extensive review covers much of the subsequent literature (5). Of course, solutes can cause nonideal behavior through a second mechanism, strong intermolecular interactions such as electrostatic and hydrophobic effects. Although crowding is now often used as a synonym for any effects of concentrated solutes, for the purpose of this commentary I will take crowding to refer to the excluded volume effects, as originally defined (3, 4), because disentangling size effects and interaction effects is precisely where the advances of Smith et al. (2) lie. I also discuss effects on equilibria only. Given our recently revised understanding of equilibrium effects, it seems premature to discuss the more complex effects of...

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“…2 persion forces. [3][4][5][6] The simplest way of accounting for a short-range solute-solvent attraction is by means of Baxter's sticky-hard-sphere (SHS) model 7 characterized by a stickiness parameter τ sl . Both issues (solvent-solvent repulsion and solute-solvent short-range attraction) were recently addressed by two experimental studies 8,9 on adsorbing microgels (MG) to large PS latex suspension.…”

Section: Introductionmentioning

confidence: 99%

Bridging and depletion mechanisms in colloid-colloid effective interactions: A reentrant phase diagram

Fantoni

Giacometti

Santos

2015

The Journal of Chemical Physics

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A general class of nonadditive sticky-hard-sphere binary mixtures, where small and large spheres represent the solvent and the solute, respectively, is introduced. The solute-solute and solvent-solvent interactions are of hard-sphere type, while the solute-solvent interactions are of sticky-hard-sphere type with tunable degrees of size nonadditivity and stickiness. Two particular and complementary limits are studied using analytical and semi-analytical tools. The first case is characterized by zero nonadditivity, lending itself to a PercusYevick approximate solution from which the impact of stickiness on the spinodal curves and on the effective solute-solute potential is analyzed. In the opposite nonadditive case, the solvent-solvent diameter is zero and the model can then be reckoned as an extension of the well-known Asakura-Oosawa model with additional sticky solute-solvent interaction. This latter model has the property that its exact effective one-component problem involves only solute-solute pair potentials for size ratios such that a solvent particle fits inside the interstitial region of three touching solutes. In particular, we explicitly identify the three competing physical mechanisms (depletion, pulling, and bridging) giving rise to the effective interaction. Some remarks on the phase diagram of these two complementary models are also addressed through the use of the Noro-Frenkel criterion and a first-order perturbation analysis. Our findings suggest reentrance of the fluid-fluid instability as solvent density (in the first model) or adhesion (in the second model) is varied. Some perspectives in terms of the interpretation of recent experimental studies of microgels adsorbed onto large polystyrene particles are discussed.

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Is the depletion force entropic? Molecular crowding beyond steric interactions

Cited by 119 publications

References 79 publications

In-cell thermodynamics and a new role for protein surfaces

In-cell thermodynamics and a new role for protein surfaces

Unpacking the origins of in-cell crowding

Bridging and depletion mechanisms in colloid-colloid effective interactions: A reentrant phase diagram

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