Unexpected Effects of Macromolecular Crowding on Protein Stability

Benton, Laura A.; Smith, Austin E.; Young, Gregory B.; Pielak, Gary J.

doi:10.1021/bi300909q

Cited by 201 publications

(234 citation statements)

References 42 publications

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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: 88%

“…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. 1 A and B. Here they examine the size dependence in more detail by comparing the effect on SH3 stability of the neutral polymers dextran, Ficoll, and PEG vs. their corresponding monomers glucose, sucrose, and ethylene glycol, respectively: chemically identical pairs of molecules of vastly different size.…”

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confidence: 99%

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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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confidence: 88%

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confidence: 99%

Unpacking the origins of in-cell crowding

Sharp

2016

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

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“…That is, an increased ΔG den o' was not always associated with a decreased ΔS den o' (and vice versa), plus the change in ΔH den o' was non-zero. As pointed out by Benton et al (2012), some of the complication observed for synthetic polymers may arise from the influence of preferential hydration (Timasheff 1993). In summary, the simplest ideas about macromolecular crowding predict entropically derived stabilization, but recent studies indicate that attractive, non-specific interactions complicate this interpretation.…”

Section: Enthalpy and Entropymentioning

confidence: 99%

“…This phenomenon has been studied theoretically and experimentally. Theoretical models have been successful in mimicking the hard-core repulsion effect (e.g., Berg 1990;Rivas et al 2001;Zhou et al 2008;McGuffee and Elcock 2010), and many 'wet' experiments show that crowding by synthetic polymers stabilizes proteins (e.g., Sasahara et al 2003;Stagg et al 2007;Charlton et al 2008;Christiansen et al 2010;Miklos et al 2010;Benton et al 2012). These observations are consistent with theoretical expectations in as much as the stabilizing hard-core repulsions seem to dominate, although significant attractive protein-crowder interactions have been noted for polyethylene glycol (Crowley et al 2008;Hirota et al 2010;Zhang et al 2012) and the monomer of polyvinylpyrrolidone .…”

Section: Crowding and Assemblymentioning

confidence: 99%

“…Independent studies using larger test proteins with both synthetic polymers and proteins as crowders have also been reported. Both Wang et al (2012), who studied his-tagged ubiquitin, and Benton et al (2012) . That is, an increased ΔG den o' was not always associated with a decreased ΔS den o' (and vice versa), plus the change in ΔH den o' was non-zero.…”

Section: Enthalpy and Entropymentioning

confidence: 99%

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Soft interactions and crowding

2013

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The intracellular milieu is complex, heterogeneous and crowded-an environment vastly different from dilute solutions in which most biophysical studies are performed. The crowded cytoplasm excludes about a third of the volume available to macromolecules in dilute solution. This excluded volume is the sum of two parts: steric repulsions and chemical interactions, also called soft interactions. Until recently, most efforts to understand crowding have focused on steric repulsions. Here, we summarize the results and conclusions from recent studies on macromolecular crowding, emphasizing the contribution of soft interactions to the equilibrium thermodynamics of protein stability. Despite their non-specific and weak nature, the large number of soft interactions present under many crowded conditions can sometimes overcome the stabilizing steric, excluded volume effect.

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Crowding interactions perturb structure and stability by destabilizing the stable core of the α‐subunit of tryptophan synthase

et al. 2016

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The consequences of crowding derived from relatively small and intrinsically disordered proteins are not clear yet. We report the effect of ficoll-70 on the structure and stability of native and partially folded states of the 29 kDa alpha subunit of tryptophan synthase (aTS). Overall, combining the changes in the circular dichroism and fluorescence spectra, in conjunction with the gradual loss of cooperativity under urea denaturation in the presence of increasing amounts of ficoll, it may be concluded that the crowding agent perturbs not only the native state but also the partially folded state of aTS. Importantly, NMR data indicate that ficoll interacts with the residues that constitute the stable core of the protein thus shedding light on the origin of the observed perturbation.

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Unexpected Effects of Macromolecular Crowding on Protein Stability

Cited by 201 publications

References 42 publications

Unpacking the origins of in-cell crowding

Unpacking the origins of in-cell crowding

Soft interactions and crowding

Crowding interactions perturb structure and stability by destabilizing the stable core of the α‐subunit of tryptophan synthase

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