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Proceedings of the National Academy of... Apr 2013Conditionally disordered proteins can alternate between highly ordered and less ordered configurations under physiological conditions. Whereas protein function is often...
Conditionally disordered proteins can alternate between highly ordered and less ordered configurations under physiological conditions. Whereas protein function is often associated with the ordered conformation, for some of these conditionally unstructured proteins, the opposite applies: Their activation is associated with their unfolding. An example is the small periplasmic chaperone HdeA, which is critical for the ability of enteric bacterial pathogens like Escherichia coli to survive passage through extremely acidic environments, such as the human stomach. At neutral pH, HdeA is a chaperone-inactive dimer. On a shift to low pH, however, HdeA monomerizes, partially unfolds, and becomes rapidly active in preventing the aggregation of substrate proteins. By mutating two aspartic acid residues predicted to be responsible for the pH-dependent monomerization of HdeA, we have succeeded in isolating an HdeA mutant that is active at neutral pH. We find this HdeA mutant to be substantially destabilized, partially unfolded, and mainly monomeric at near-neutral pH at a concentration at which it prevents aggregation of a substrate protein. These results provide convincing evidence for direct activation of a protein by partial unfolding.
Topics: Amino Acid Sequence; Base Sequence; Circular Dichroism; Escherichia coli Proteins; Hydrogen-Ion Concentration; Models, Molecular; Molecular Chaperones; Molecular Dynamics Simulation; Molecular Sequence Data; Mutagenesis; Periplasm; Protein Binding; Protein Conformation; Protein Unfolding; Sequence Alignment; Sequence Analysis, DNA; Ultracentrifugation
PubMed: 23487787
DOI: 10.1073/pnas.1222458110 -
Journal of Bacteriology Mar 2005
Review
Topics: Bacteria; Drug Resistance, Multiple, Bacterial; Membrane Transport Proteins; Periplasm
PubMed: 15743933
DOI: 10.1128/JB.187.6.1879-1883.2005 -
Journal of Molecular Biology Jan 2018HdeA is a periplasmic chaperone that is rapidly activated upon shifting the pH to acidic conditions. This activation is thought to involve monomerization of HdeA. There...
HdeA is a periplasmic chaperone that is rapidly activated upon shifting the pH to acidic conditions. This activation is thought to involve monomerization of HdeA. There is evidence that monomerization and partial unfolding allow the chaperone to bind to proteins denatured by low pH, thereby protecting them from aggregation. We analyzed the acid-induced unfolding of HdeA using NMR spectroscopy and fluorescence measurements, and obtained experimental evidence suggesting a complex mechanism in HdeA's acid-induced unfolding pathway, as previously postulated from molecular dynamics simulations. Counterintuitively, dissociation constant measurements show a stabilization of the HdeA dimer upon exposure to mildly acidic conditions. We provide experimental evidence that protonation of Glu37, a glutamate residue embedded in a hydrophobic pocket of HdeA, is important in controlling HdeA stabilization and thus the acid activation of this chaperone. Our data also reveal a sharp transition from folded dimer to unfolded monomer between pH3 and pH 2, and suggest the existence of a low-populated, partially folded intermediate that could assist in chaperone activation or function. Overall, this study provides a detailed experimental investigation into the mechanism by which HdeA unfolds and activates.
Topics: Escherichia coli; Escherichia coli Proteins; Glutamic Acid; Hydrogen-Ion Concentration; Molecular Chaperones; Molecular Dynamics Simulation; Periplasm; Protein Conformation; Protein Denaturation; Protein Folding; Protein Unfolding
PubMed: 29138002
DOI: 10.1016/j.jmb.2017.11.002 -
Philosophical Transactions of the Royal... Apr 2012Autotransport in Gram-negative bacteria denotes the ability of surface-localized proteins to cross the outer membrane (OM) autonomously. Autotransporters perform this... (Review)
Review
Autotransport in Gram-negative bacteria denotes the ability of surface-localized proteins to cross the outer membrane (OM) autonomously. Autotransporters perform this task with the help of a β-barrel transmembrane domain localized in the OM. Different classes of autotransporters have been investigated in detail in recent years; classical monomeric but also trimeric autotransporters comprise many important bacterial virulence factors. So do the two-partner secretion systems, which are a special case as the transported protein resides on a different polypeptide chain than the transporter. Despite the great interest in these proteins, the exact mechanism of the transport process remains elusive. Moreover, different periplasmic and OM factors have been identified that play a role in the translocation, making the term 'autotransport' debatable. In this review, we compile the wealth of details known on the mechanism of single autotransporters from different classes and organisms, and put them into a bigger perspective. We also discuss recently discovered or rediscovered classes of autotransporters.
Topics: Adhesins, Bacterial; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Secretion Systems; Cell Membrane; Gram-Negative Bacteria; Models, Molecular; Periplasm; Protein Folding; Protein Structure, Tertiary; Protein Transport; Virulence Factors
PubMed: 22411980
DOI: 10.1098/rstb.2011.0208 -
Future Microbiology Aug 2013
Topics: Bacteria; Coenzymes; Periplasm; Protein Binding; Zinc; beta-Lactam Resistance; beta-Lactams
PubMed: 23902139
DOI: 10.2217/fmb.13.34 -
ELife Apr 2022Surface lipoproteins (SLPs) are peripherally attached to the outer leaflet of the outer membrane in many Gram-negative bacteria, playing significant roles in nutrient...
Surface lipoproteins (SLPs) are peripherally attached to the outer leaflet of the outer membrane in many Gram-negative bacteria, playing significant roles in nutrient acquisition and immune evasion in the host. While the factors that are involved in the synthesis and delivery of SLPs in the inner membrane are well characterized, the molecular machinery required for the movement of SLPs to the surface are still not fully elucidated. In this study, we investigated the translocation of a SLP TbpB through a Slam1-dependent pathway. Using purified components, we developed an in vitro translocation assay where unfolded TbpB is transported through Slam1-containing proteoliposomes, confirming Slam1 as an outer membrane translocon. While looking to identify factors to increase translocation efficiency, we discovered the periplasmic chaperone Skp interacted with TbpB in the periplasm of . The presence of Skp was found to increase the translocation efficiency of TbpB in the reconstituted translocation assays. A knockout of Skp in revealed that Skp is essential for functional translocation of TbpB to the bacterial surface. Taken together, we propose a pathway for surface destined lipoproteins, where Skp acts as a holdase for Slam-mediated TbpB translocation across the outer membrane.
Topics: Bacterial Outer Membrane Proteins; Escherichia coli; Escherichia coli Proteins; Lipoproteins; Periplasm
PubMed: 35475756
DOI: 10.7554/eLife.72822 -
Microbiology (Reading, England) Jun 2019Rhizobium etli CE3 grown in succinate-ammonium minimal medium (MM) excreted outer membrane vesicles (OMVs) with diameters of 40 to 100 nm. Proteins from the OMVs and...
Rhizobium etli CE3 grown in succinate-ammonium minimal medium (MM) excreted outer membrane vesicles (OMVs) with diameters of 40 to 100 nm. Proteins from the OMVs and the periplasmic space were isolated from 6 and 24 h cultures and identified by proteome analysis. A total of 770 proteins were identified: 73.8 and 21.3 % of these occurred only in the periplasm and OMVs, respectively, and only 4.9 % were found in both locations. The majority of proteins found in either location were present only at 6 or 24 h: in the periplasm and OMVs, only 24 and 9 % of proteins, respectively, were present at both sampling times, indicating a time-dependent differential sorting of proteins into the two compartments. The OMVs contained proteins with physiologically varied roles, including Rhizobium adhering proteins (Rap), polysaccharidases, polysaccharide export proteins, auto-aggregation and adherence proteins, glycosyl transferases, peptidoglycan binding and cross-linking enzymes, potential cell wall-modifying enzymes, porins, multidrug efflux RND family proteins, ABC transporter proteins and heat shock proteins. As expected, proteins with known periplasmic localizations (phosphatases, phosphodiesterases, pyrophosphatases) were found only in the periplasm, along with numerous proteins involved in amino acid and carbohydrate metabolism and transport. Nearly one-quarter of the proteins present in the OMVs were also found in our previous analysis of the R. etli total exproteome of MM-grown cells, indicating that these nanoparticles are an important mechanism for protein excretion in this species.
Topics: Bacterial Proteins; Culture Media; Extracellular Vesicles; Periplasm; Proteome; Rhizobium etli
PubMed: 30358529
DOI: 10.1099/mic.0.000720 -
Biophysical Journal Jan 2011The physical and mechanical properties of the cell envelope of Escherichia coli are poorly understood. We use fluorescence recovery after photobleaching to measure...
The physical and mechanical properties of the cell envelope of Escherichia coli are poorly understood. We use fluorescence recovery after photobleaching to measure diffusion of periplasmic green fluorescent protein and probe the fluidity of the periplasm as a function of external osmotic conditions. For cells adapted to growth in complete medium at 0.14-1.02 Osm, the mean diffusion coefficient
increases from 3.4 μm² s⁻¹ to 6.6 μm² s⁻¹ and the distribution of D(peri) broadens as growth osmolality increases. This is consistent with a net gain of water by the periplasm, decreasing its biopolymer volume fraction. This supports a model in which the turgor pressure drops primarily across the thin peptidoglycan layer while the cell actively maintains osmotic balance between periplasm and cytoplasm, thus avoiding a substantial pressure differential across the cytoplasmic membrane. After sudden hyperosmotic shock (plasmolysis), the cytoplasm loses water as the periplasm gains water. Accordingly, increases threefold. The fluorescence recovery after photobleaching is complete and homogeneous in all cases, but in minimal medium, the periplasm is evidently thicker at the cell tips. For the relevant geometries, Brownian dynamics simulations in model cytoplasmic and periplasmic volumes provide analytical formulae for extraction of accurate diffusion coefficients from readily measurable quantities. Topics: Culture Media; Cytoplasm; Diffusion; Escherichia coli; Fluorescence Recovery After Photobleaching; Green Fluorescent Proteins; Microscopy, Fluorescence; Osmotic Pressure; Periplasm; Protein Transport
PubMed: 21190653
DOI: 10.1016/j.bpj.2010.11.044 -
BioTechniques Feb 2023Single-chain variable fragments (ScFvs) are important in therapy, diagnosis and research because of their elevated antigen affinity and low immunogenicity. At present,...
Single-chain variable fragments (ScFvs) are important in therapy, diagnosis and research because of their elevated antigen affinity and low immunogenicity. At present, high-yield scFv expression in is limited by insoluble aggregation in the reducing environment of the cytoplasm or low yields in the periplasm. Here we achieved increased expression of scFvs in the periplasm by inserting optimal amino acids between the signal peptide and scFv. We constructed an expression library with three random amino acids at the scFv N-terminus, screened this library with a single-step colony assay and identified the specific sequences that boosted periplasmic expression of scFvs.
Topics: Single-Chain Antibodies; Escherichia coli; Periplasm
PubMed: 36748400
DOI: 10.2144/btn-2022-0107 -
Scientific Reports Oct 2017The major outer sheath protein (MOSP) is a prominent constituent of the cell envelope of Treponema denticola (TDE) and one of its principal virulence determinants....
The major outer sheath protein (MOSP) is a prominent constituent of the cell envelope of Treponema denticola (TDE) and one of its principal virulence determinants. Bioinformatics predicts that MOSP consists of N- and C-terminal domains, MOSP and MOSP. Biophysical analysis of constructs refolded in vitro demonstrated that MOSP, previously shown to possess porin activity, forms amphiphilic trimers, while MOSP forms an extended hydrophilic monomer. In TDE and E. coli expressing MOSP with a PelB signal sequence (PelB-MOSP), MOSP is OM-embedded and surface-exposed, while MOSP resides in the periplasm. Immunofluorescence assay, surface proteolysis, and novel cell fractionation schemes revealed that MOSP in TDE exists as outer membrane (OM) and periplasmic trimeric conformers; PelB-MOSP, in contrast, formed only OM-MOSP trimers. Although both conformers form hetero-oligomeric complexes in TDE, only OM-MOSP associates with dentilisin. Mass spectrometry (MS) indicated that OM-MOSP interacts with proteins in addition to dentilisin, most notably, oligopeptide-binding proteins (OBPs) and the β-barrel of BamA. MS also identified candidate partners for periplasmic MOSP, including TDE1658, a spirochete-specific SurA/PrsA ortholog. Collectively, our data suggest that MOSP destined for the TDE OM follows the canonical BAM pathway, while formation of a stable periplasmic conformer involves an export-related, folding pathway not present in E. coli.
Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; Escherichia coli; Escherichia coli Proteins; Mass Spectrometry; Periplasm; Treponema denticola
PubMed: 29038532
DOI: 10.1038/s41598-017-13550-6