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The Journal of Biological Chemistry May 2012The Escherichia coli NhaA antiporter couples the transport of H(+) and Na(+) (or Li(+)) ions to maintain the proper pH range and Na(+) concentration in cells. A crystal...
The Escherichia coli NhaA antiporter couples the transport of H(+) and Na(+) (or Li(+)) ions to maintain the proper pH range and Na(+) concentration in cells. A crystal structure of NhaA, solved at pH 4, comprises 12 transmembrane helices (TMs), arranged in two domains, with a large cytoplasm-facing funnel and a smaller periplasm-facing funnel. NhaA undergoes conformational changes, e.g. after pH elevation to alkaline ranges, and we used two computational approaches to explore them. On the basis of pseudo-symmetric features of the crystal structure, we predicted the structural architecture of an alternate, periplasm-facing state. In contrast to the crystal structure, the model presents a closed cytoplasmic funnel, and a periplasmic funnel of greater volume. To examine the transporter functional direction of motion, we conducted elastic network analysis of the crystal structure and detected two main normal modes of motion. Notably, both analyses predicted similar trends of conformational changes, consisting of an overall rotational motion of the two domains around a putative symmetry axis at the funnel centers, perpendicular to the membrane plane. This motion, along with conformational changes within specific helices, resulted in closure at the cytoplasmic end and opening at the periplasmic end. Cross-linking experiments, performed between segments on opposite sides of the cytoplasmic funnel, revealed pH-dependent interactions consistent with the proposed conformational changes. We suggest that the model-structure and predicted motion represent alkaline pH-induced conformational changes, mediated by a cluster of evolutionarily conserved, titratable residues, at the cytoplasmic ends of TMs II, V, and IX.
Topics: Escherichia coli Proteins; Hydrogen-Ion Concentration; Models, Molecular; Periplasm; Protein Conformation; Sodium-Hydrogen Exchangers
PubMed: 22431724
DOI: 10.1074/jbc.M111.336446 -
Proceedings of the National Academy of... Mar 2022SignificanceHow flagella sense complex environments and control bacterial motility remain fascinating questions. Here, we deploy cryo-electron tomography to determine in...
SignificanceHow flagella sense complex environments and control bacterial motility remain fascinating questions. Here, we deploy cryo-electron tomography to determine in situ structures of the flagellar motor in wild-type and mutant cells of , revealing that three flagellar proteins (FliL, MotA, and MotB) form a unique supramolecular complex in situ. Importantly, FliL not only enhances motor function by forming a ring around the stator complex MotA/MotB in its extended, active conformation but also facilitates assembly of the stator complex around the motor. Our in situ data provide insights into how cooperative remodeling of the FliL-stator supramolecular complex helps regulate the collective ion flux and establishes the optimal function of the flagellar motor to guide bacterial motility in various environments.
Topics: Bacterial Physiological Phenomena; Bacterial Proteins; Borrelia burgdorferi; Flagella; Gene Expression Regulation, Bacterial; Membrane Proteins; Models, Biological; Models, Molecular; Molecular Motor Proteins; Periplasm
PubMed: 35254893
DOI: 10.1073/pnas.2117245119 -
Methods in Enzymology 2016Artificial metalloenzymes represent an attractive means of combining state-of-the-art transition metal catalysis with the benefits of natural enzymes. Despite the...
Artificial metalloenzymes represent an attractive means of combining state-of-the-art transition metal catalysis with the benefits of natural enzymes. Despite the tremendous recent progress in this field, current efforts toward the directed evolution of these hybrid biocatalysts mainly rely on the laborious, individual purification of protein variants rendering the throughput, and hence the outcome of these campaigns feeble. We have recently developed a screening platform for the directed evolution of artificial metalloenzymes based on the streptavidin-biotin technology in the periplasm of the Gram-negative bacterium Escherichia coli. This periplasmic compartmentalization strategy comprises a number of compelling advantages, in particular with respect to artificial metalloenzymes, which lead to a drastic increase in the throughput of screening campaigns and additionally are of unique value for future in vivo applications. Therefore, we highlight here the benefits of this strategy and intend to propose a generalized guideline for the development of novel transition metal-based biocatalysts by directed evolution in order to extend the natural enzymatic repertoire.
Topics: Catalysis; Directed Molecular Evolution; Enzymes; Metalloproteins; Metals; Periplasm; Protein Engineering
PubMed: 27586348
DOI: 10.1016/bs.mie.2016.05.037 -
BioMed Research International 2019All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable... (Review)
Review
All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an "off" to an "on" state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.
Topics: Biosensing Techniques; Periplasm; Periplasmic Binding Proteins; Protein Domains; Protein Engineering
PubMed: 30719443
DOI: 10.1155/2019/4798793 -
Environmental Microbiology Reports Oct 2015This review emphasizes the biological roles of the osmoregulated periplasmic glucans (OPGs). Osmoregulated periplasmic glucans occur in almost all α-, β- and... (Review)
Review
This review emphasizes the biological roles of the osmoregulated periplasmic glucans (OPGs). Osmoregulated periplasmic glucans occur in almost all α-, β- and γ-Proteobacteria. This polymer of glucose is required for full virulence. The roles of the OPGs are complex and vary depending on the species. Here, we outline the four major roles of the OPGs through four different pathogenic and one symbiotic bacterial models (Dickeya dadantii, Salmonella enterica, Pseudomonas aeruginosa, Brucella abortus and Sinorhizobium meliloti). When periplasmic, the OPGs are a part of the signal transduction pathway and indirectly regulate genes involved in virulence. The OPGs can also be secreted. When outside of the cell, they interact directly with antibiotics to protect the bacterial cell or interact with the host cell to facilitate the invasion process. When OPGs are not found, as in the ε-Proteobacteria, OPG-like oligosaccharides are present. Their presence strengthens the evidence that OPGs play an important role in virulence.
Topics: Alphaproteobacteria; Anti-Bacterial Agents; Gammaproteobacteria; Glucans; Osmoregulation; Periplasm; Signal Transduction; Virulence
PubMed: 26265506
DOI: 10.1111/1758-2229.12325 -
Biochimie 2002The aim of this review is to describe an in vivo assay of the interactions taking place in the Tol-Pal or TonB-ExbB-ExbD envelope complexes in the periplasm of... (Review)
Review
The aim of this review is to describe an in vivo assay of the interactions taking place in the Tol-Pal or TonB-ExbB-ExbD envelope complexes in the periplasm of Escherichia coli and between them and colicins or g3p protein of filamentous bacteriophages. Domains of colicins or periplasmic soluble domains of Tol or TonB proteins can be artificially addressed to the periplasm of bacteria by fusing them to a signal sequence from an exported protein. These domains interact specifically in the periplasm with the Tol or TonB complexes and disturb their function, which can be directly detected by the appearance of specific tol or tonB phenotypes. This technique can be used to detect new interactions, to characterize them biochemically and to map them or to induce tol or tonB phenotypes to study the functions of these two complexes.
Topics: Bacterial Proteins; Capsid Proteins; Colicins; DNA-Binding Proteins; Escherichia coli; Escherichia coli Proteins; Membrane Proteins; Periplasm; Viral Fusion Proteins
PubMed: 12423784
DOI: 10.1016/s0300-9084(02)01423-2 -
Proceedings of the National Academy of... May 2022The Porphyromonas gingivalis type IX secretion system (T9SS) promotes periodontal disease by secreting gingipains and other virulence factors. By in situ cryoelectron...
The Porphyromonas gingivalis type IX secretion system (T9SS) promotes periodontal disease by secreting gingipains and other virulence factors. By in situ cryoelectron tomography, we report that the P. gingivalis T9SS consists of 18 PorM dimers arranged as a large, caged ring in the periplasm. Near the outer membrane, PorM dimers interact with a PorKN ring complex of ∼52 nm in diameter. PorMKN translocation complexes of a given T9SS adopt distinct conformations energized by the proton motive force, suggestive of different activation states. At the inner membrane, PorM associates with a cytoplasmic complex that exhibits 12-fold symmetry and requires both PorM and PorL for assembly. Activated motors deliver substrates across the outer membrane via one of eight Sov translocons arranged in a ring. The T9SSs are unique among known secretion systems in bacteria and eukaryotes in their assembly as supramolecular machines composed of apparently independently functioning translocation motors and export pores.
Topics: Bacterial Proteins; Bacterial Secretion Systems; Periplasm; Porphyromonas gingivalis; Virulence Factors
PubMed: 35471908
DOI: 10.1073/pnas.2119907119 -
FEBS Letters May 1998Genetic studies have recently identified DsbG, a new member of the dsb group of redox proteins, which catalyze protein disulfide bond formation in the periplasm of...
Genetic studies have recently identified DsbG, a new member of the dsb group of redox proteins, which catalyze protein disulfide bond formation in the periplasm of Escherichia coli. We now demonstrate that DsbG functions primarily as an oxidant during protein disulfide bond formation, which is consistent with the low stability of its active site disulfide bond. There are indications, however, that the substrate range of DsbG may be narrower than the other periplasmic oxidative enzymes, DsbA and DsbC. Our observations further elaborate the pathway of disulfide bond formation in E. coli.
Topics: Aprotinin; Escherichia coli; Escherichia coli Proteins; Kinetics; Models, Chemical; Oxidation-Reduction; Oxidoreductases; Periplasm; Periplasmic Proteins; Protein Folding; Recombinant Proteins
PubMed: 9654144
DOI: 10.1016/s0014-5793(98)00539-0 -
Biotechnology Journal Jul 2017A high cell density fed-batch process was developed for production of recombinant CRM197, a non-toxic mutant of diphtheria toxin widely used as a carrier in...
A high cell density fed-batch process was developed for production of recombinant CRM197, a non-toxic mutant of diphtheria toxin widely used as a carrier in polysaccharide-protein conjugate vaccines. Fully soluble recombinant CRM197 was obtained in high yields and with an authentic N-terminus, by targeting the protein to the periplasm of Escherichia coli using the Signal Recognition Particle (SRP)-dependent signal sequence of FlgI. Response Surface Methodology (RSM) was used to optimize the set-points of key process parameters (pH and feed rate at induction). Optimal production of periplasmic CRM197 was found at a slightly basic pH (7.5). The feed rate during induction was positively correlated with the accumulation of unprocessed cytoplasmic CRM197, consistent with limited capacity of the SRP secretion pathway. Decreasing the feed rate to align the protein synthesis rate with the secretion capacity, resulted in minimal production of cytoplasmic CRM197. Besides, the host background was found critical for production of periplasmic CRM197: B834(DE3) was the highest producer (>3 g/L), while BLR(DE3) produced one third less CRM197, and very low yields (290 mg/L) were obtained with HMS174(DE3). The optimized process is robust and linearly scalable, and represents a 20-fold yield improvement compared to a process based on Corynebacterium diphtheriae.
Topics: Bacterial Proteins; Batch Cell Culture Techniques; Escherichia coli; Fermentation; Hydrogen-Ion Concentration; Periplasm; Signal Recognition Particle
PubMed: 28397983
DOI: 10.1002/biot.201700168 -
Microbiology (Reading, England) Sep 2022Neutrophilic Fe(II) oxidizing bacteria play an important role in biogeochemical processes and have also received attention for multiple technological applications. These...
Neutrophilic Fe(II) oxidizing bacteria play an important role in biogeochemical processes and have also received attention for multiple technological applications. These micro-organisms are thought to couple their metabolism with extracellular electron transfer (EET) while oxidizing Fe(II) as electron donor outside the cell. ES-1 is a freshwater chemolithoautotrophic Fe(II) oxidizing bacterium that is challenging to culture and not yet genetically tractable. Analysis of the ES-1 genome predicts multiple EET pathways, which are proposed to be involved in Fe(II) oxidation, but not yet validated. Here we expressed components of two of the proposed EET pathways, including the Mto and Slit_0867-0870 PCC3 pathways from ES-1 into , an established model EET organism. We demonstrate that combinations of putative inner membrane and periplasmic components from the Mto and Slit_0867-0870 PCC3 pathways partially complemented EET activity in mutants lacking native components. Our results provide evidence for electron transfer functionality and interactions of inner membrane and periplasmic components from the Mto and Slit_0867-0870 PCC3 pathways. Based on these findings, we suggest that EET in ES-1 could be more complicated than previously considered and raises questions regarding directionality of these electron transfer pathways.
Topics: Electron Transport; Electrons; Ferrous Compounds; Oxidation-Reduction; Periplasm
PubMed: 36111788
DOI: 10.1099/mic.0.001240