Pressure‐ and heat‐induced protein unfolding in bacterial cells: crowding <i>vs</i>. sticking

Chen, Timothy; Dave, Kapil; Gruebele, Martin

doi:10.1002/1873-3468.13025

Cited by 18 publications

(18 citation statements)

References 32 publications

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“…Finally, we anticipate that the effect of hydrostatic pressure on protein compaction and folding is negligible under our conditions because the pressure necessary to unfold PGK-FRET is >600 bar. 52 Therefore, cytoskeletal drugs can produce two competing crowding effects inside cells: depolymerizing the cytoskeleton decreases crowder size and increases the fluidity of the cytoplasm, whereas reduced cell volume increases the extent of crowding.…”

Section: ■ Resultsmentioning

confidence: 99%

Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells

Davis

Gruebele

2020

Biochemistry

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The dynamic cytoskeletal network of microtubules and actin filaments can be disassembled by drugs. Cytoskeletal drugs work by perturbing the monomer–polymer equilibrium, thus changing the size and number of macromolecular crowders inside cells. Changes in both crowding and nonspecific surface interactions (“sticking”) following cytoskeleton disassembly can affect the protein stability, structure, and function directly or indirectly by changing the fluidity of the cytoplasm and altering the crowding and sticking of other macromolecules in the cytoplasm. The effect of cytoskeleton disassembly on protein energy landscapes inside cells has yet to be observed. Here we have measured the effect of several cytoskeletal drugs on the folding energy landscape of two FRET-labeled proteins with different in vitro sensitivities to macromolecular crowding. Phosphoglycerate kinase (PGK) was previously shown to be more sensitive to crowding, whereas variable major protein-like sequence expressed (VlsE) was previously shown to be more sensitive to sticking. The in-cell effects of drugs that depolymerize either actin filaments (cytochalasin D and latrunculin B) or microtubules (nocodazole and vinblastine) were compared. The crowding sensor protein CrH2-FRET verified that cytoskeletal drugs decrease the extent of crowding inside cells despite also reducing the overall cell volume. The decreased compactness and folding stability of PGK could be explained by the decreased extent of crowding induced by these drugs. VlsE’s opposite response to the drugs shows that depolymerization of the cytoskeleton also changes sticking in the cellular milieu. Our results demonstrate that perturbation of the monomer–polymer cytoskeletal equilibrium, for example, during natural cell migration or stresses from drug treatment, has off-target effects on the energy landscapes of proteins in the cell.

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Section: ■ Resultsmentioning

confidence: 99%

Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells

Davis

Gruebele

2020

Biochemistry

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“…Other recent work has shown that a eukaryotic probe protein introduced into E . coli will be less destabilized by pressure than temperature denaturation in E. coli , relative to in vitro pressure and temperature denaturation results [33]. Thus, destabilization of the protein by ‘sticking’ to the cytoplasmic matrix is mitigated by high pressure (better crowding), but enhanced by high temperature (stronger hydrophobic effect [34]).…”

Section: Folding Phase Diagrams Under Extreme Conditionsmentioning

confidence: 99%

Protein folding and surface interaction phase diagrams in vitro and in cells

Gruebele

2021

FEBS Letters

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Protein stability is subject to environmental perturbations such as pressure and crowding, as well as sticking to other macromolecules and quinary structure. Thus, the environment inside and outside the cell plays a key role in how proteins fold, interact, and function on the scale from a few molecules to macroscopic ensembles. This review discusses three aspects of protein phase diagrams: first, the relevance of phase diagrams to protein folding and function in vitro and in cells; next, how the evolution of protein surfaces impacts on interaction phase diagrams; and finally, how phase separation plays a role on much larger length‐scales than individual proteins or oligomers, when liquid phase‐separated regions form to assist protein function and cell homeostasis.

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“…In addition to their role in the spatial arrangement of the cellular milieu, quinary interactions modulate protein folding and stability . In‐cell studies showed that quinary interactions can have both stabilising and destabilising effects on globular proteins. The role of these opposite effects is still not clear but preliminary evidences suggests a dependence on protein sequence, function and location .…”

Section: Intracellular Spatial Organisation: the Emerging Role Of Quimentioning

confidence: 99%

“…These interactions, also known as nonsteric or soft interactions, are those that a protein can perform with its surroundings beyond the steric interactions that result from the excluded volume effect . They stabilise or destabilise globular proteins through charge–charge, polar, hydrogen bond and hydrophobic contacts with both the native and unfolded states (for more details see Appendix 1) . The magnitude of this modulation ranges on average from 0.1 to 1.1 kcal·mol −1 .…”

Section: Physicochemical Properties Of the Intracellular Environment mentioning

confidence: 99%

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Protein folding and quinary interactions: creating cellular organisation through functional disorder

2018

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The marginal stability of globular proteins in the cell is determined by the balance between excluded volume effect and soft interactions. Quinary interactions are a type of soft interactions involved in intracellular organisation and known to have stabilising or destabilising effects on globular proteins. Recent studies suggest that globular proteins have structural flexibility, exhibiting more than one functional state. Here, we propose that the quinary-induced destabilisation can be sufficient to produce functional partially unfolded states of globular proteins. The biological relevance of this mechanism is explored, involving intracellular phase separation and regulatory stress response mechanisms.

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Pressure‐ and heat‐induced protein unfolding in bacterial cells: crowding vs. sticking

Cited by 18 publications

References 32 publications

Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells

Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells

Protein folding and surface interaction phase diagrams in vitro and in cells

Protein folding and quinary interactions: creating cellular organisation through functional disorder

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