Exploring the dynamics and structure of PpiB in living Escherichia coli cells using electron paramagnetic resonance spectroscopy

Protein Science, Journal Year: 2024, Volume and Issue: 33(3)

Published: Feb. 15, 2024

The combined effects of the cellular environment on proteins led to definition a fifth level protein structural organization termed quinary structure. To explore implication potential structure for globular proteins, we studied dynamics and conformations Escherichia coli (E. coli) peptidyl-prolyl cis/trans isomerase B (PpiB) in E. cells. PpiB plays major role maturation regulation folded by catalyzing isomerization proline imidic peptide bond. We applied electron paramagnetic resonance (EPR) techniques, utilizing both Gadolinium (Gd(III)) nitroxide spin labels. In addition using standard labeling approaches with genetically engineered cysteines, incorporated an unnatural amino acid achieve Gd(III)-nitroxide orthogonal labeling. probed PpiB's residue-specific X-band continuous wave EPR at ambient temperatures its double electron-electron (DEER) frozen samples. was delivered cells electroporation. report significant decrease induced two chosen positions. These changes could not be reproduced adding crowding agents cell extracts. Concomitantly, broadening distance distribution coli, determined Gd(III)-Gd(III) DEER measurements, as compared solution human HeLa This suggests increase number present cells, possibly due interactions other components, which also contributes reduction mobility presence

Language: Английский

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Robustness and Sensitivity of Gd(III)–Gd(III) Double Electron–Electron Resonance (DEER) Measurements: Comparative Study of High-Frequency EPR Spectrometer Designs and Spin Label Variants

Applied Magnetic Resonance, Journal Year: 2025, Volume and Issue: 56(5), P. 591 - 611

Published: Jan. 3, 2025

Abstract In this paper, we explore the robustness and sensitivity of Gd(III)-Gd(III) double electron–electron resonance (DEER) distance measurements in proteins for different spectrometer designs three spin labels. To do a protein was labeled at same two positions with Gd(III) labels were performed on home-built high-frequency (W-band, ~ 95 GHz) EPR spectrometers design approaches, commercial 150 W Q-band (34 spectrometer. The first W-band measurement approach uses conventional, narrow band single mode cavity, while second broadband non-resonant induction sample holder. Both systems incorporate advanced arbitrary waveform generators (AWGs) that give flexibility over excitation bandwidth. We use DOTA-like labels, Gd.C12, Gd.DO3A Gd.L 1 , conjugated to calmodulin protein. compare taken by including or excluding central transition excitation. advantages disadvantages Gd(III)–Gd(III) DEER are discussed terms resulting distribution width, absolute concentration sensitivity, handling, ease use, measurement.

Language: Английский

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Phosphate Limitation Enhances Heterologous Enzyme Production in Bacillus subtilis: Mechanistic Insights and Universal Applicability

Journal of Agricultural and Food Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 29, 2025

Bacillus subtilis is one of the commonly used hosts for heterologous enzyme expression, depending on media rich in carbon, nitrogen, and phosphate sources optimal growth production. Interestingly, our investigation maltotetraose-forming amylase, a key efficient maltotetraose synthesis, revealed that limitation significantly enhances rate production enzymes recombinant B. subtilis. Under phosphate-limited conditions 15 L fermenter, activity reached 679.15 U/mL, an improvement 101% over initial levels 12 h reduction fermentation time. Transcriptomic analysis indicated promotes sustained by upregulating protein synthesis quality control pathways while optimizing energy utilization. This strategy was validated across various systems, highlighting its general applicability enhancing expressions. These findings provide valuable insights industrial amylase other high-value enzymes, supporting advancement microbial technology.

Language: Английский

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Characterization of conformationally heterogeneous proteins by electron paramagnetic resonance spectroscopy

Current Opinion in Structural Biology, Journal Year: 2025, Volume and Issue: 92, P. 103046 - 103046

Published: April 11, 2025

Language: Английский

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Dynamic basis of lipopolysaccharide export by LptB2FGC

eLife, Journal Year: 2024, Volume and Issue: 13

Published: July 1, 2024

Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB 2 FG forms an ATP-binding cassette transporter that transfers to LptC. How couples ATP binding and hydrolysis with LptC remains unclear. We observed conformational heterogeneity FGC micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored release laser-induced liquid bead ion desorption mass spectrometry. β-jellyroll domain LptF stably interacts LptG β-jellyrolls both apo vanadate-trapped states. at cytoplasmic side is allosterically coupled selective opening periplasmic domain. In FG, closes nucleotide domains, causing a collapse first lateral gate as structures. However, second gate, which putative entry site for LPS, exhibits heterogeneous conformation. limits flexibility this two conformations, likely representing helix either released from or inserted into transmembrane domains. Our results reveal regulation through dynamic behavior helix, while its anchored periplasm. This, combined long-range ATP-dependent allosteric gating domain, may ensure efficient unidirectional

Language: Английский

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Endogenous Cu(II) Labeling for Distance Measurements on Proteins by EPR

Chemistry - A European Journal, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 14, 2024

In-cell measurements of the relationship between structure and dynamics to protein function is at forefront biophysics. Recently, developments in EPR methodology have demonstrated sensitivity power this method measure structural constraints in-cell. However, need spin label proteins ex-situ or use noncanonical amino acids achieve endogenous labeling remains a bottleneck. In work we expand endogenously with Cu(II) labels describe how assess in-cell labeling. We quantify amount Cu(II)-NTA cells, labeling, account for orientational effects during distance measurements. compare efficacy using heat-shock hypotonic swelling deliver label, showing that facile reproducible efficiently into E. coli. Notably, over six repeats accomplish bulk average 57 μM labeled sites, surpassing existing methods. The results open door easily accessible broader biophysical community.

Language: Английский

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Protein‐protein interaction and conformational change in the alpha‐helical membrane transporter BtuCD‐F in the native cellular envelope

ChemBioChem, Journal Year: 2024, Volume and Issue: 26(1)

Published: Nov. 18, 2024

Abstract Alpha‐helical membrane proteins perform numerous critical functions essential for the survival of living organisms. Traditionally, these are extracted from membranes using detergent solubilization and reconstitution into liposomes or nanodiscs. However, processes often obscure effects nanoconfinement native environment on structure conformational heterogeneity target protein. We demonstrate that pulsed dipolar electron spin resonance spectroscopy, combined with Gd 3+ ‐nitroxide pair, enables selective observation vitamin B 12 importer BtuCD−F in its cellular envelope. Despite high levels non‐specific labeling envelope, this orthogonal approach long phase‐memory time protein complex at a few micromolar concentrations resolution. In induces distinct shift BtuCD‐BtuF interface, which is not observed micelles. This offers general strategy investigating protein‐protein protein‐ligand/drug interactions changes alpha‐helical their envelope context.

Language: Английский

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Dynamic basis of lipopolysaccharide export by LptB₂FGC

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: May 21, 2024

Abstract Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB 2 FG forms an ATP-binding cassette transporter that transfers to LptC. How couples ATP binding and hydrolysis with LptC remains unclear. We observed conformational heterogeneity FGC micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored release laser-induced liquid bead ion desorption mass spectrometry. β-jellyroll domain LptF stably interacts LptG β-jellyrolls both apo vanadate-trapped states. at cytoplasmic side is allosterically coupled selective opening periplasmic domain. In FG, closes nucleotide domains, causing a collapse first lateral gate as structures. However, second gate, which putative en try site for LPS, exhibits heterogeneous conformation. limits flexibility this two conformations, likely representing helix either released from or inserted into transmembrane domains. Our results reveal regulation entry through dynamic behavior helix, while its anchored periplasm. This, combined long-range ATP-dependent allosteric gating domain, may ensure efficient unidirectional

Language: Английский

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Dynamic basis of lipopolysaccharide export by LptB2FGC

Published: July 1, 2024

Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB 2 FG forms an ATP-binding cassette transporter that transfers to LptC. How couples ATP binding and hydrolysis with LptC remains unclear. We observed conformational heterogeneity FGC micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored release laser-induced liquid bead ion desorption mass spectrometry. β-jellyroll domain LptF stably interacts LptG β-jellyrolls both apo vanadate-trapped states. at cytoplasmic side is allosterically coupled selective opening periplasmic domain. In FG, closes nucleotide domains, causing a collapse first lateral gate as structures. However, second gate, which putative en try site for LPS, exhibits heterogeneous conformation. limits flexibility this two conformations, likely representing helix either released from or inserted into transmembrane domains. Our results reveal regulation entry through dynamic behavior helix, while its anchored periplasm. This, combined long-range ATP-dependent allosteric gating domain, may ensure efficient unidirectional

Language: Английский

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Dynamic basis of lipopolysaccharide export by LptB2FGC

Published: Sept. 2, 2024

Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB2FG forms an ATP-binding cassette transporter that transfers to LptC. How couples ATP binding and hydrolysis with LptC remains unclear. We observed conformational heterogeneity LptB2FGC micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored release laser-induced liquid bead ion desorption mass spectrometry. β-jellyroll domain LptF stably interacts LptG β-jellyrolls both apo vanadate-trapped states. at cytoplasmic side is allosterically coupled selective opening periplasmic domain. In LptB2FG, closes nucleotide domains, causing a collapse first lateral gate as structures. However, second gate, which putative entry site for LPS, exhibits heterogeneous conformation. limits flexibility this two conformations, likely representing helix either released from or inserted into transmembrane domains. Our results reveal regulation through dynamic behavior helix, while its anchored periplasm. This, combined long-range ATP-dependent allosteric gating domain, may ensure efficient unidirectional

Language: Английский

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