A FRET-based method for monitoring structural transitions in protein self-organization

Wan, Qi; Mouton, Sara N.; Veenhoff, Liesbeth M.; Boersma, Arnold J.

doi:10.1016/j.crmeth.2022.100184

Cited by 10 publications

(13 citation statements)

References 44 publications

(60 reference statements)

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“…35 We obtain insight into the protein self-assembly behavior of H-NS using intermolecular FRET. 36 We see that the macromolecular crowding enhances the oligomerization of H-NS in both dilute lysate and in E. coli , although the H-NS oligomerization in E. coli requires time to respond to a sudden crowding change.…”

Section: Introductionmentioning

confidence: 84%

“…37 We recently showed that this method allows straightforward determination of protein self-organization in a precise and high-throughput manner. 36 The method relies on a tight fusion between an mVenus and mCherry (VC) with low intramolecular FRET due to the restricted motion between the two fluorophores. In contrast, the formation of condensates, aggregates, and oligomers should increase FRET efficiency through intermolecular FRET.…”

Section: In Vitro H-ns Self-association Is Sensitive To Salts Crowdin...mentioning

confidence: 99%

“…In contrast, the formation of condensates, aggregates, and oligomers should increase FRET efficiency through intermolecular FRET. 36,38 The readout depends on the intermolecular distance between the FRET pairs fused to the target proteins, which in turn is a function of the density and structure in the assembly. 38,39 We fused H-NS to the VC domain via a short 5-Glycine linker (Figure 1).…”

Section: In Vitro H-ns Self-association Is Sensitive To Salts Crowdin...mentioning

confidence: 99%

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Self-association of a nucleoid-binding protein increases with macromolecular crowding inEscherichia coli

Pittas

Boersma

2023

Preprint

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Many proteins self-associate to achieve function. Macromolecular crowding enhances protein self-assembly in buffer experiments with added crowders, and crowding could therefore regulate protein function and organization in cells. In eukaryotic cells, protein condensation has been shown to increase with crowding. However, it is unclear what the effect of crowding is on native protein self-assembly in the highly crowded Escherichia coli cell. To determine the role of crowding in the self-assembly of a native protein, we study here the nucleoid-binding H-NS in E. coli and alter macromolecular crowding using a set of perturbations. We followed H-NS self-assembly using a FRET-based method for determining intermolecular interactions with a single genetic intervention. In dilute cell lysate, we see that H-NS self-assembly increases with salts, macromolecular crowding, and its own concentration. In E. coli, the oligomerization increases with crowding. We see that the response of H-NS oligomerization to a sudden crowding change is not immediate but requires time to adapt. Our findings implicate that in-cell crowding affects intracellular organization by promoting self-assembly.

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Section: Introductionmentioning

confidence: 84%

Section: In Vitro H-ns Self-association Is Sensitive To Salts Crowdin...mentioning

confidence: 99%

Section: In Vitro H-ns Self-association Is Sensitive To Salts Crowdin...mentioning

confidence: 99%

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Self-association of a nucleoid-binding protein increases with macromolecular crowding inEscherichia coli

Pittas

Boersma

2023

Preprint

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“…During the decrease in crowding effects, the OD increases slowly (Figure 2d). To completely exclude optical or fluorescent protein artifacts in these stress conditions, we tested a mVenus-mCherry construct without a linker 37 to prevent intramolecular FRET changes. We found that the FRET ratios of spheroplasted and Penicillin-treated cells are the same as exponentially growing cells (Figure 2e).…”

Section: Cell Wall Damage Causes a Macromolecular Crowding Increasementioning

confidence: 99%

“…We used E. coli BL21(DE3) for every experiment. The synthetic genes crGE2.3 53 and VC 37 in the plasmid pRSET-A were codon-optimized for E. coli and obtained from GeneArt. Bacteria were transformed with crGE2.3 (meGFP-mScarlet-I) or the crGE (mCerulean3-mCitrine) crowding sensor.…”

Section: Growth Conditions Of E Coli Cellsmentioning

confidence: 99%

Cell wall damage increases macromolecular crowding effects in the Escherichia coli cytoplasm

Pittas

Zuo

Boersma

2022

Preprint

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The intracellular milieu is crowded with biomacromolecules. Macromolecular crowding changes the interactions, diffusion, and conformations of the biomacromolecules. Changes in intracellular crowding effects have been mostly ascribed to differences in biomacromolecule concentration. However, the spatial organization of these molecules should play a significant role in crowding effects. Here, we find that cell wall damage causes increased macromolecular crowding effects in the Escherichia coli cytoplasm. Using a genetically-encoded macromolecular crowding sensor, we see that crowding effects in E. coli spheroplasts and Penicillin G-treated cells well surpass crowding effects obtained using hyperosmotic stress. The crowding increase is not due to osmotic pressure, cell shape, crowder synthesis, or volume changes, and therefore not crowder concentration. Instead, a genetically-encoded nucleic acid stain and a small molecule DNA stain show nucleoid expansion and cytoplasmic mixing, which could cause these increased crowding effects. Our data demonstrate that cell stress from antibiotics or cell wall damage alters the biochemical organization in the cytoplasm and induces significant conformational changes in a probe protein.

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Advanced imaging techniques for studying protein phase separation in living cells and at single-molecule level

Mekonnen

Djaja

Yuan

et al. 2023

Current Opinion in Chemical Biology

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A FRET-based method for monitoring structural transitions in protein self-organization

Cited by 10 publications

References 44 publications

Self-association of a nucleoid-binding protein increases with macromolecular crowding inEscherichia coli

Self-association of a nucleoid-binding protein increases with macromolecular crowding inEscherichia coli

Cell wall damage increases macromolecular crowding effects in the Escherichia coli cytoplasm

Advanced imaging techniques for studying protein phase separation in living cells and at single-molecule level

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