Recombinant Proteus mirabilis ATP synthase subunit c (atpE)

Shipped with Ice Packs
In Stock

Description

Introduction to Recombinant Proteus mirabilis ATP Synthase Subunit c (atpE)

Recombinant Proteus mirabilis ATP synthase subunit c (atpE) is a bioengineered protein derived from the F-type ATP synthase complex of Proteus mirabilis (strain HI4320). ATP synthase subunit c is a critical transmembrane component of the Fo/Vo complex, forming part of the c-ring rotor that drives proton translocation and ATP synthesis in bacteria . In Proteus mirabilis, this subunit is encoded by the atpE gene (Uniprot ID: B4F0E2) and plays a central role in energy metabolism .

Functional Mechanism

  1. Proton Translocation: Subunit c’s glutamic acid (Glu) or aspartic acid (Asp) residues (e.g., Asp61 in Escherichia coli) undergo sequential protonation/deprotonation, enabling stepwise rotor rotation .

  2. ATP Synthesis Coupling: The c-ring’s rotation is mechanically linked to the F1 domain’s catalytic β-subunits, driving ATP synthesis from ADP and inorganic phosphate .

Antibiotic Resistance Studies

In Proteus mirabilis, ATP synthase subunit c has been indirectly linked to polymyxin resistance. A study identified a gene with similarity to ATP synthase subunits in polymyxin-resistant mutants, suggesting potential roles in membrane potential regulation . While not directly tested in Proteus mirabilis, ATP synthase inhibition in other pathogens (e.g., Staphylococcus aureus) hyperpolarizes membranes, increasing susceptibility to polymyxins .

Biofilm Formation and Infection Models

ATP synthase subunit c has been targeted in other bacteria (e.g., Streptococcus mutans) to inhibit biofilm formation and acid production, highlighting its potential as a therapeutic target .

ELISA-Based Research Tools

Recombinant Proteus mirabilis atpE is commercially available as an ELISA antigen for detecting specific antibodies or studying immune responses .

Production and Availability

Recombinant atpE is typically expressed in heterologous systems (e.g., E. coli) with optimized protocols for solubility and purification . Key production parameters include:

ParameterDetail
Expression HostE. coli (commonly used for bacterial recombinant proteins)
Purity>90% (determined by SDS-PAGE)
Storage-20°C or -80°C (avoid repeated freeze-thaw cycles)

Product Specs

Form
Lyophilized powder
Note: While we prioritize shipping the format currently in stock, we are happy to accommodate special requests for the format. Please specify your preferred format during the order placement, and we will prepare accordingly.
Lead Time
Delivery times may vary depending on the purchasing method and location. We recommend consulting your local distributors for specific delivery time estimates.
Note: All protein shipments are standardly delivered with blue ice packs. If dry ice shipping is required, please inform us in advance for additional fees.
Notes
Repeated freezing and thawing is not recommended. For optimal usage, store working aliquots at 4°C for up to one week.
Reconstitution
Prior to opening, we suggest briefly centrifuging the vial to gather the contents at the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, we recommend adding 5-50% glycerol (final concentration) and aliquoting at -20°C/-80°C. Our standard final glycerol concentration is 50% and can be used as a reference.
Shelf Life
The shelf life of our proteins is influenced by several factors, including storage conditions, buffer ingredients, storage temperature, and the inherent stability of the protein.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Storage Condition
Upon receipt, store at -20°C/-80°C. For multiple use, aliquoting is essential. Avoid repeated freeze-thaw cycles.
Tag Info
Tag type is determined during the manufacturing process.
We determine the tag type during the production process. If you have a specific tag type requirement, please inform us and we will prioritize developing the specified tag.
Synonyms
atpE; PMI3059; ATP synthase subunit c; ATP synthase F(0 sector subunit c; F-type ATPase subunit c; F-ATPase subunit c; Lipid-binding protein
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
1-79
Protein Length
full length protein
Species
Proteus mirabilis (strain HI4320)
Target Names
atpE
Target Protein Sequence
MENLSMDLLYMAAAIMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLV DAIPMIAVGLGLYVMFAVA
Uniprot No.

Target Background

Function
F(1)F(0) ATP synthase generates ATP from ADP in the presence of a proton or sodium gradient. F-type ATPases consist of two structural domains: F(1) containing the extramembraneous catalytic core and F(0) containing the membrane proton channel, connected by a central stalk and a peripheral stalk. During catalysis, ATP synthesis in the catalytic domain of F(1) is coupled to proton translocation via a rotary mechanism of the central stalk subunits. As a key component of the F(0) channel, the c-ring plays a direct role in translocation across the membrane. A homomeric c-ring of between 10-14 subunits forms the central stalk rotor element with the F(1) delta and epsilon subunits.
Database Links

KEGG: pmr:PMI3059

STRING: 529507.PMI3059

Protein Families
ATPase C chain family
Subcellular Location
Cell inner membrane; Multi-pass membrane protein.

Q&A

What is the role of ATP synthase subunit c (AtpE) in Proteus mirabilis metabolism?

ATP synthase subunit c is an essential enzyme component that catalyzes ATP production from ADP in the presence of sodium or proton gradients. In P. mirabilis, this enzyme plays a critical role in energy production, particularly during various growth phases and environmental conditions. The protein forms part of the FO domain of ATP synthase, specifically functioning within the membrane proton channel. The c-subunits form the homomeric c-ring (comprising 10-14 subunits) that serves as the central rotor element of the F1 domain. Key residues between positions 5-25 and 57-77 constitute the functional FO domain, which is vital for the rotary mechanism during catalysis .

How does P. mirabilis AtpE structure compare to homologous proteins in other bacterial species?

Analysis of homology modeling reveals that P. mirabilis AtpE shares significant structural features with ATP synthase subunit c from other bacterial species. While specific data for P. mirabilis AtpE is limited in the search results, comparable studies on Mycobacterium tuberculosis AtpE demonstrated that high sequence similarity (>90%) between homologous proteins typically results in highly conserved structures with RMSD values below 0.6 Å after structural superimposition . Researchers working with P. mirabilis AtpE should perform similar template-based homology modeling using closely related structures from the Protein Data Bank, followed by energy minimization and refinement via molecular dynamics simulation to generate reliable structural models.

What expression systems are most effective for producing recombinant P. mirabilis AtpE?

For recombinant expression of membrane proteins like AtpE, E. coli-based expression systems typically provide good yields when optimized properly. The methodology involves:

  • Gene synthesis or PCR amplification of the atpE gene from P. mirabilis genomic DNA

  • Cloning into an expression vector with an appropriate promoter (T7 or tac)

  • Expression in E. coli strains designed for membrane protein production (C41(DE3), C43(DE3), or Lemo21(DE3))

  • Growth at lower temperatures (16-25°C) after induction to facilitate proper folding

  • Extraction using mild detergents like n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG)

This methodological approach maintains protein structural integrity while maximizing yield for downstream applications such as crystallography or inhibitor screening.

How can molecular docking studies identify potential inhibitors of P. mirabilis AtpE?

Molecular docking studies for AtpE inhibitor identification require a structured methodological approach:

  • Generate a refined 3D model structure of P. mirabilis AtpE using homology modeling based on templates with high sequence identity

  • Perform energy minimization and refinement using molecular dynamic simulation (10 ns is typical) with tools like AMBERTOOLS10

  • Validate model quality using Ramachandran plots, ERRAT, and Verify_3D

  • Select a diverse compound library (ZINC or PubChem databases are recommended)

  • Perform virtual screening using tools like RASPD and PyRx to identify compounds with favorable binding energies

  • Filter compounds using Lipinski's rule of five parameters:

    • Molecular weight ≤500 Da

    • LogP ≤5

    • Hydrogen bond donors ≤5

    • Hydrogen bond acceptors ≤10

The docking protocol should utilize appropriate grid dimensions (60 × 60 × 60 Å with 0.375 Å spacing has proven effective) and employ Lamarckian genetic algorithms for binding energy calculations .

What structural features distinguish the ATP binding site of P. mirabilis AtpE from human ATP synthase?

While P. mirabilis AtpE and human ATP synthase subunit c share conserved functionality, subtle structural differences in their ATP binding sites make AtpE a potential antimicrobial target. The binding site analysis methodology involves:

  • Superposition of bacterial and human ATP synthase structural models

  • Identification of residue variations in the binding pocket

  • Characterization of electrostatic potential differences using tools like APBS (Adaptive Poisson-Boltzmann Solver)

  • Analysis of hydrophobic/hydrophilic property variations

These subtle differences can be exploited for selective inhibitor design, as demonstrated in similar analyses of ATP synthase from other bacterial species. This approach has potential for developing antimicrobials with reduced human toxicity for treating P. mirabilis infections, particularly catheter-associated urinary tract infections (CAUTIs) .

How does P. mirabilis AtpE function change during biofilm formation in catheter-associated infections?

P. mirabilis is a significant causative agent in CAUTIs, with biofilm formation being a critical virulence mechanism . During biofilm development on catheter surfaces, P. mirabilis undergoes metabolic adaptations that likely affect AtpE function. Research methodologies to investigate this include:

  • Comparative transcriptomics and proteomics of planktonic versus biofilm P. mirabilis cells

  • ATP production assays under varied environmental conditions mimicking the urinary catheter microenvironment

  • Site-directed mutagenesis of key AtpE residues to assess functional impacts on biofilm formation

  • Correlation analysis between AtpE activity and other virulence factors like urease, which is constitutively expressed during growth in urine

Understanding these adaptations may identify novel approaches for preventing biofilm formation on catheter surfaces.

What purification strategy provides optimal yield and purity for recombinant P. mirabilis AtpE?

The methodology for optimal purification of recombinant P. mirabilis AtpE follows a structured protocol:

  • Extraction from expression host using buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% detergent (typically DDM)

  • Initial purification via Immobilized Metal Affinity Chromatography (IMAC) using a histidine tag

  • Secondary purification via size exclusion chromatography (SEC) to remove aggregates and improve homogeneity

  • Quality assessment using SDS-PAGE, mass spectrometry, and circular dichroism to confirm identity and proper folding

For structural studies requiring higher purity:

  • Consider additional ion exchange chromatography step

  • Remove fusion tags using specific proteases (TEV or thrombin)

  • Concentrate using ultrafiltration devices with appropriate molecular weight cutoffs

This methodological approach typically yields >95% pure protein suitable for functional and structural studies.

How can researchers effectively measure ATP synthase activity of recombinant P. mirabilis AtpE?

ATP synthase activity measurement requires reconstitution of the functional enzyme complex. The methodological approach includes:

  • Reconstitution of purified AtpE with other ATP synthase subunits in liposomes

  • Generation of proton gradient using appropriate buffer systems

  • Measurement of ATP production using luciferase-based luminescence assays or coupled enzyme assays

  • Data analysis accounting for background ATP hydrolysis

For inhibition studies, researchers should:

  • Pre-incubate the reconstituted enzyme with test compounds

  • Use dose-response curves to determine IC50 values

  • Apply Michaelis-Menten kinetics to determine inhibition mechanisms

This approach allows quantitative assessment of functional activity and provides a platform for inhibitor screening.

What computational methods best predict the impact of mutations on P. mirabilis AtpE function?

Computational prediction of mutation effects requires a multi-faceted approach:

  • Generate a refined structural model of P. mirabilis AtpE via homology modeling

  • Introduce mutations using computational mutagenesis tools

  • Perform energy minimization of mutant structures

  • Conduct molecular dynamics simulations (minimum 10 ns) to assess structural stability

  • Calculate binding free energy differences using Molecular Mechanics Generalized Born and Surface Area (MM-GBSA) analysis

  • Analyze changes in:

    • Protein stability using FoldX or Rosetta

    • Hydrogen bonding patterns

    • Electrostatic interactions

    • Hydrophobic packing

These computational predictions should be validated experimentally using site-directed mutagenesis and functional assays.

How should researchers resolve discrepancies in AtpE inhibitor screening data across different methodologies?

When confronted with conflicting inhibitor screening results, researchers should implement a systematic resolution approach:

  • Compare experimental conditions across studies:

    • Buffer composition and pH

    • Protein concentration and purity

    • Incubation time and temperature

    • Detection methods

  • Validate hits using orthogonal assays:

    • Thermal shift assays to confirm binding

    • Surface plasmon resonance (SPR) to determine binding kinetics

    • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Perform control experiments with known inhibitors/substrates

  • Consider the impact of detergents or lipids on assay performance

  • Analyze structure-activity relationships to identify consistent patterns despite quantitative differences

This methodological approach helps distinguish true biological differences from technical artifacts and strengthens the reliability of screening results.

What factors should be considered when interpreting MD simulation data for AtpE-inhibitor complexes?

Molecular dynamics simulation data interpretation requires careful consideration of several factors:

The table below summarizes typical parameter ranges for stable AtpE-inhibitor complexes based on similar studies:

These guidelines help distinguish meaningful biological interactions from simulation artifacts.

How can researchers differentiate between effects specific to AtpE inhibition versus general membrane disruption?

Distinguishing specific AtpE inhibition from general membrane effects requires a multi-assay approach:

  • Implement control experiments:

    • Test compounds against liposomes without AtpE to detect membrane disruption

    • Measure effects on membrane potential using fluorescent probes (DiSC3(5))

    • Assess general cytotoxicity using mammalian cell lines

  • Perform structure-activity relationship (SAR) studies:

    • Synthesize analogs with varying physicochemical properties

    • Correlate structural changes with activity and membrane effects

  • Use site-directed mutagenesis:

    • Introduce mutations at predicted binding sites

    • Resistance mutations confer specificity to AtpE-targeted inhibition

  • Compare with known membrane disruptors and specific ATP synthase inhibitors

This methodological framework provides strong evidence for mechanism of action discrimination.

How might heterologous expression systems be optimized for structural studies of P. mirabilis AtpE?

Optimization of heterologous expression for structural studies requires addressing several challenges:

  • Selection of appropriate expression hosts:

    • E. coli C41(DE3) or C43(DE3) for initial screening

    • Insect cell systems for eukaryotic processing if needed

    • Cell-free systems for toxic proteins

  • Vector design optimization:

    • Fusion partners (SUMO, MBP) to enhance solubility

    • Inclusion of purification tags at positions verified not to disrupt function

    • Codon optimization for expression host

  • Cultivation condition optimization:

    • Induction at lower temperatures (16-20°C)

    • Extended expression times (24-48 hours)

    • Specialized media formulations

  • Detergent screening for extraction and purification:

    • Systematic testing of detergent types and concentrations

    • Nanodiscs or SMALPs for maintaining native lipid environment

This systematic approach maximizes the likelihood of obtaining protein suitable for high-resolution structural studies via X-ray crystallography or cryo-electron microscopy.

What novel approaches might be developed to study AtpE interactions with other components of the ATP synthase complex?

Advanced methodologies for studying AtpE interactions include:

  • In situ proximity labeling techniques:

    • BioID or APEX2 fusion constructs to identify interacting proteins

    • Crosslinking mass spectrometry (XL-MS) to map specific interaction interfaces

  • Single-molecule techniques:

    • Förster resonance energy transfer (FRET) to measure dynamic interactions

    • High-speed atomic force microscopy to visualize conformational changes

  • Computational approaches:

    • Molecular docking guided by evolutionary coupling analysis

    • Coarse-grained molecular dynamics simulations of the entire ATP synthase complex

  • Genetic approaches:

    • Suppressor mutation analysis to identify functional interactions

    • Bacterial two-hybrid systems adapted for membrane protein interactions

These complementary approaches provide a comprehensive understanding of AtpE's role within the complex ATP synthase machinery.

Quick Inquiry

Personal Email Detected
Please use an institutional or corporate email address for inquiries. Personal email accounts ( such as Gmail, Yahoo, and Outlook) are not accepted. *
© Copyright 2025 TheBiotek. All Rights Reserved.