Large cosolutes, small cosolutes, and dihydrofolate reductase activity

Acosta, Luis C.; Goncalves, Gerardo M. Perez; Pielak, Gary J.; Gorensek-Benitez, Annelise H.

doi:10.1002/pro.3316

Cited by 34 publications

(45 citation statements)

References 78 publications

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“…These limitations do not, however, change the fact that the hard-core component has a larger effect on the domain-swapped dimer. We did not apply the theory to the synthetic polymers, because these macromolecules cannot be accurately modeled as spheres, because, at the high concentrations used here (39)(40)(41), the individual polymer molecules overlap to form a mesh (42). The reason is exemplified by the concentration dependence of viscosity.…”

Section: Discussionmentioning

confidence: 99%

Protein shape modulates crowding effects

Guseman

Goncalves

Speer

et al. 2018

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

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Protein−protein interactions are usually studied in dilute buffered solutions with macromolecule concentrations of <10 g/L. In cells, however, the macromolecule concentration can exceed 300 g/L, resulting in nonspecific interactions between macromolecules. These interactions can be divided into hard-core steric repulsions and "soft" chemical interactions. Here, we test a hypothesis from scaled particle theory; the influence of hard-core repulsions on a protein dimer depends on its shape. We tested the idea using a side-by-side dumbbell-shaped dimer and a domain-swapped ellipsoidal dimer. Both dimers are variants of the B1 domain of protein G and differ by only three residues. The results from the relatively inert synthetic polymer crowding molecules, Ficoll and PEG, support the hypothesis, indicating that the domain-swapped dimer is stabilized by hard-core repulsions while the side-by-side dimer shows little to no stabilization. We also show that protein cosolutes, which interact primarily through nonspecific chemical interactions, have the same small effect on both dimers. Our results suggest that the shape of the protein dimer determines the influence of hard-core repulsions, providing cells with a mechanism for regulating protein−protein interactions. macromolecular crowding | protein−protein interactions | scaled particle theory P rotein−protein interactions are essential for maintaining cellular homeostasis (1). Details of their equilibria under thermodynamically ideal conditions have provided a trove of information. Ideality in this sense refers to dilute solutions, where each monomer contacts only solvent or another monomer, conditions far removed from those in cells where protein− protein interactions evolved. In the cytoplasm, and other cellular compartments and biological fluids, macromolecules can occupy up to 30% of the volume, and their concentrations often exceed 300 g/L (2).Protein molecules take part in more-complex interactions under nonideal conditions. The surrounding macromolecules influence proteins in two ways, neither of which is significant in dilute solution. Hard-core repulsions arise from high volume occupancy, because two molecules cannot occupy the same space at the same time. This volume exclusion favors the most compact state of a protein (3). Chemical interactions comprise transient contacts between protein surfaces arising from the diverse chemical landscapes of proteins (4). When repulsive (i.e., like charges), they favor the state that maximizes the distance between charges, adding to the hard-core repulsions and stabilizing the native state. Attractive chemical interactions (e.g., opposite charges, hydrogen bonds) are destabilizing. We are beginning to understand how hard-core repulsions and chemical interactions affect protein stability (5), but there are few studies about crowding effects on protein−protein interactions (6-9).Given the existential roles of both protein−protein interactions and crowding in biology (10), we are undertaking efforts to determine the effect of cro...

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Section: Discussionmentioning

confidence: 99%

Protein shape modulates crowding effects

Guseman

Goncalves

Speer

et al. 2018

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

Self Cite

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“…Inert macromolecules such as dextran, Ficoll, polyethylene glycol (PEG) or proteins (e. g. albumin) are commonly used to mimic cellular crowding in vitro . These experiments have demonstrated that crowding stabilizes proteins, modulates enzymatic activity,, and enhances association …”

Section: Figurementioning

confidence: 99%

Non‐Steric Interactions Predict the Trend and Steric Interactions the Offset of Protein Stability in Cells

Davis

Gruebele

2018

ChemPhysChem

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Although biomolecules evolved to function in the cell, most biochemical assays are carried out in vitro. In-cell studies highlight how steric and non-steric interactions modulate protein folding and interactions. VlsE and PGK present two extremes of chemical behavior in the cell: the extracellular protein VlsE is destabilized in eukaryotic cells, whereas the cytoplasmic protein PGK is stabilized. VlsE and PGK are benchmarks in a systematic series of solvation environments to distinguish contributions from non-steric and steric interactions to protein stability, compactness, and folding rate by comparing cell lysate, a crowding agent, ionic buffer and lysate buffer with in-cell results. As anticipated, crowding stabilizes proteins, causes compaction, and can speed folding. Protein flexibility determines its sensitivity to steric interactions or crowding. Non-steric interactions alone predict in-cell stability trends, while crowding provides an offset towards greater stabilization. We suggest that a simple combination of lysis buffer and Ficoll is an effective new in vitro mimic of the intracellular environment on protein folding and stability.

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“…3 To mimic cellular crowding in vitro, inert macromolecules such as dextran, Ficoll, polyethylene glycol (PEG) or proteins (e.g., albumin) can be added to the buffer. 4 In vitro studies with inert macromolecules added to the buffer demonstrate that crowding compacts and stabilizes proteins, 5,6 modulates enzymatic activity, 7,8 and enhances association. 9 Yet crowding alone cannot account for all interactions inside cells; Superoxide dismutase, 10 variable major protein-like sequence expressed (VlsE), 11,12 B1 domain of protein G (GB1), 13 and chymotrypsin inhibitor 2 (Cl2) 14 all are destabilized inside cells compared to in vitro.…”

Section: Introductionmentioning

confidence: 99%

An in vitro mimic of in‐cell solvation for protein folding studies

2020

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Ficoll, an inert macromolecule, is a common in vitro crowder, but by itself it does not reproduce in‐cell stability or kinetic trends for protein folding. Lysis buffer, which contains ions, glycerol as a simple kosmotrope, and mimics small crowders with hydrophilic/hydrophobic patches, can reproduce sticking trends observed in cells but not the crowding. We previously suggested that the proper combination of Ficoll and lysis buffer could reproduce the opposite in‐cell folding stability trend of two proteins: variable major protein‐like sequence expressed (VlsE) is destabilized in eukaryotic cells and phosphoglycerate kinase (PGK) is stabilized. Here, to discover a well‐characterized solvation environment that mimics in‐cell stabilities for these two very differently behaved proteins, we conduct a two‐dimensional scan of Ficoll (0–250 mg/ml) and lysis buffer (0–75%) mixtures. Contrary to our previous expectation, we show that mixtures of Ficoll and lysis buffer have a significant nonadditive effect on the folding stability. Lysis buffer enhances the stabilizing effect of Ficoll on PGK and inhibits the stabilizing effect of Ficoll on VlsE. We demonstrate that a combination of 150 mg/ml Ficoll and 60% lysis buffer can be used as an in vitro mimic to account for both crowding and non‐steric effects on PGK and VlsE stability and folding kinetics in the cell. Our results also suggest that this mixture is close to the point where phase separation will occur. The simple mixture proposed here, based on commercially available reagents, could be a useful tool to study a variety of cytoplasmic protein interactions, such as folding, binding and assembly, and enzymatic reactions. Significance Statement The complexity of the in‐cell environment is difficult to reproduce in the test tube. Here we validate a mimic of cellular crowding and sticking interactions in a test tube using two proteins that are differently impacted by the cell: one is stabilized and the other is destabilized. This mimic is a starting point to reproduce cellular effects on a variety of protein and biomolecular interactions, such as folding and binding.

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Large cosolutes, small cosolutes, and dihydrofolate reductase activity

Cited by 34 publications

References 78 publications

Protein shape modulates crowding effects

Protein shape modulates crowding effects

Non‐Steric Interactions Predict the Trend and Steric Interactions the Offset of Protein Stability in Cells

An in vitro mimic of in‐cell solvation for protein folding studies

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