Recombinant Pelobacter propionicus ATP synthase subunit c 2 (atpE2)

What is Pelobacter propionicus ATP synthase subunit c 2 (atpE2) and what is its role in bacterial energy metabolism?

ATP synthase subunit c 2 (atpE2) is a critical component of the F0 sector of the ATP synthase complex in Pelobacter propionicus. This protein forms part of the c-ring structure in the membrane domain of ATP synthase, which is essential for proton translocation across the bacterial membrane. The c-ring works in concert with other subunits to harness the energy from proton movement to drive ATP synthesis from ADP and inorganic phosphate. In the catalytic mechanism, protons bind to conserved glutamate residues in the c-subunits, causing rotation of the c-ring, which in turn drives conformational changes in the F1 sector to synthesize ATP .

The atpE2 protein belongs to a family of ATP synthase subunit c proteins with the UniProt ID A1AP46 and is also known by several synonyms including Ppro_1501, ATP synthase F(0) sector subunit c 2, F-type ATPase subunit c 2, and Lipid-binding protein 2 . Understanding the structure and function of this subunit is vital for comprehending bacterial bioenergetics and potential antimicrobial target identification.

How does the c-ring interface with other components of the ATP synthase complex?

The c-ring in bacterial ATP synthases like that of Pelobacter propionicus interfaces with several other components to form a functional complex. Most critically, it interacts with subunit a, which provides the half-channels for proton access to the c-ring's glutamate residues. This interaction creates two distinct proton half-channels: a periplasmic half-channel that allows protons to enter from the periplasm and a cytoplasmic half-channel that allows protons to exit to the cytoplasm .

During ATP synthesis, protons travel through the periplasmic half-channel to bind to the conserved glutamate residue of a c-subunit. This protonation event allows the c-ring to rotate counter-clockwise (when viewed from F1 toward F0), moving the protonated subunit into the hydrophobic lipid bilayer. As rotation continues, the protonated glutamate eventually reaches the cytoplasmic half-channel, where interaction with a positively charged arginine residue (Arg 169 in Bacillus PS3, equivalent to Arg 210 in E. coli) promotes proton release to the cytoplasm .

This rotary mechanism translates the energy of the proton motive force into mechanical rotation of the central stalk, which then drives conformational changes in the F1 sector catalytic sites to synthesize ATP. The entire process represents one of nature's most elegant molecular machines for energy conversion.

What are the optimal conditions for recombinant expression of Pelobacter propionicus atpE2?

For successful recombinant expression of Pelobacter propionicus ATP synthase subunit c 2 (atpE2), E. coli has proven to be an effective heterologous host system. The protein can be expressed with an N-terminal His-tag to facilitate purification . For optimal expression, consider the following protocol:

• Vector selection: Use a vector with a strong, inducible promoter (such as T7) and appropriate antibiotic resistance.

• Host strain: BL21(DE3) or its derivatives are recommended for membrane protein expression.

• Culture conditions:

  • Initial growth at 37°C to OD600 of 0.6-0.8

  • Temperature reduction to 18-20°C before induction

  • Induction with 0.1-0.5 mM IPTG

  • Post-induction growth for 16-20 hours

• Media optimization:

  • For unlabeled protein: 2xYT or TB media

  • For isotope labeling (NMR studies): M9 minimal media with appropriate isotopes

When working with membrane proteins like atpE2, it's crucial to optimize detergent conditions during both expression and purification phases. The hydrophobic nature of this protein necessitates careful handling to maintain its structural integrity and function.

What purification strategies are most effective for isolating high-purity atpE2 protein?

Purification of recombinant Pelobacter propionicus atpE2 requires special considerations due to its hydrophobic nature. The following multi-step purification strategy is recommended:

• Cell lysis: Use mechanical disruption (French press or sonication) in buffer containing protease inhibitors.

• Membrane preparation:

  • Differential centrifugation (low-speed centrifugation to remove unbroken cells, followed by high-speed ultracentrifugation to collect membranes)

  • Membrane washing to remove peripheral proteins

• Solubilization:

  • Use mild detergents such as DDM (n-dodecyl β-D-maltoside) or LMNG (lauryl maltose neopentyl glycol)

  • Typical concentration: 1% detergent in buffer containing 150-300 mM NaCl, 50 mM Tris-HCl pH 8.0

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Binding buffer: 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.05% selected detergent, 20 mM imidazole

  • Washing: Increasing imidazole concentrations (40-60 mM)

  • Elution: 250-300 mM imidazole

• Size exclusion chromatography:

  • Further purification using Superdex 200

  • Buffer: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% selected detergent

The typical purity achieved should be greater than 90% as determined by SDS-PAGE . For functional studies, it's crucial to ensure that the protein remains properly folded in its native conformation throughout the purification process.

How can researchers functionally reconstitute Pelobacter propionicus ATP synthase for in vitro studies?

Functional reconstitution of ATP synthase components into liposomes provides a powerful approach for studying their bioenergetic properties. For Pelobacter propionicus atpE2, the following reconstitution protocol is recommended:

• Liposome preparation:

  • Prepare liposomes using E. coli polar lipids or a defined mixture (e.g., POPC:POPE:cardiolipin at 7:2:1 ratio)

  • Dissolve lipids in chloroform, evaporate solvent to form lipid film

  • Hydrate with reconstitution buffer (typically 10 mM MOPS pH 7.5, 100 mM KCl)

  • Sonicate or extrude to form unilamellar vesicles of uniform size

• Protein incorporation:

  • Add detergent-solubilized atpE2 protein to liposomes at protein:lipid ratios of 1:50 to 1:100 (w/w)

  • Incubate for 30 minutes at room temperature

  • Remove detergent using Bio-Beads or dialysis

• Assembly with other ATP synthase components:

  • For minimal functional studies, co-reconstitute with purified subunit a and other F0 components

  • For complete ATP synthesis studies, include the F1 sector (α3β3γδε)

• Functional assays:

  • ATP synthesis: Establish proton gradient with acid-base transition

  • Proton pumping: Monitor pH changes with ACMA fluorescence quenching

  • ATP hydrolysis: Enzyme-coupled assay measuring phosphate release

For optimal results, it's critical to verify incorporation and orientation of the protein in liposomes, as randomized orientation can compromise functional measurements .

What methods can be used to study proton translocation through reconstituted c-rings?

Proton translocation through c-rings is central to ATP synthase function. For studying this process in reconstituted systems containing Pelobacter propionicus atpE2, several complementary approaches can be employed:

• pH-sensitive fluorescent probes:

  • ACMA (9-amino-6-chloro-2-methoxyacridine): Quenching indicates proton uptake into liposomes

  • pyranine: Ratiometric measurements for quantitative pH determination

  • Experimental setup: Add probes during or after liposome reconstitution; establish baseline; add substrate (e.g., ATP for hydrolysis mode); observe fluorescence changes

• Membrane potential-sensitive dyes:

  • DiSC3(5) (3,3'-dipropylthiadicarbocyanine iodide): Indicates membrane potential formation

  • Oxonol VI: Alternative for membrane potential measurements

• SSM-based electrophysiology (Solid-supported membrane):

  • Direct measurement of charge translocation

  • Allows time-resolved measurements of proton pumping activity

• Isotope exchange assays:

  • H2^18O/H2^16O exchange to track proton movement

• Site-directed mutagenesis:

  • Strategic mutation of key residues (e.g., conserved glutamate) to confirm their role

These methods can be combined with biochemical assays that measure ATP synthesis or hydrolysis to correlate proton movement with enzymatic activity. For instance, researchers have shown that bacterial ATP synthases can generate up to two ATP molecules per H2 oxidized in optimized systems .

What structural features distinguish Pelobacter propionicus atpE2 from other bacterial c-subunits?

Pelobacter propionicus atpE2 shares core structural features with other bacterial ATP synthase c-subunits while possessing distinctive characteristics. Analysis of its 91-amino acid sequence reveals:

The hydrophobic profile of atpE2 is characteristic of membrane-embedded c-subunits, with approximately 70% hydrophobic residues . The c-subunit must form precise interactions with neighboring subunits in the c-ring, as well as with subunit a, to create the proper environment for proton translocation.

Structural comparisons with well-characterized bacterial c-subunits (such as those from E. coli and Bacillus PS3) can provide valuable insights into the evolutionary adaptations of Pelobacter propionicus ATP synthase to its specific environmental niche.

What experimental approaches are most informative for structural studies of atpE2?

Multiple complementary approaches can be employed for structural investigations of Pelobacter propionicus atpE2:

• X-ray crystallography:

  • Requires highly purified, detergent-solubilized protein

  • Crystallization screening in lipidic cubic phases often successful for membrane proteins

  • Can provide atomic-level details of structure

• Cryo-electron microscopy (cryo-EM):

  • Particularly useful for intact ATP synthase complexes

  • Sample preparation: vitrification of purified protein in detergent or reconstituted in nanodiscs

  • Modern direct electron detectors enable near-atomic resolution

  • Has revealed critical structural features of bacterial ATP synthases, including the arrangement of c-ring subunits and their interaction with subunit a

• NMR spectroscopy:

  • Solution NMR for detergent-solubilized protein

  • Solid-state NMR for membrane-embedded studies

  • Especially informative for dynamics and protonation states

• Molecular dynamics simulations:

  • Complement experimental data

  • Provide insights into proton movement and conformational changes

  • Require experimental structures as starting points

• Cross-linking mass spectrometry:

  • Identifies interaction interfaces with other ATP synthase subunits

  • Chemical or photo-crosslinking followed by MS analysis

Cryo-EM has proven particularly valuable for recent structural studies of bacterial ATP synthases, revealing the architecture of the proton translocation pathway through the membrane domain. These structures show how conserved residues create two half-channels at the interface of subunit a and the c-ring, providing a path for protons to access the critical glutamate residues .

How can atpE2 be utilized in synthetic biology applications for ATP production?

Recombinant Pelobacter propionicus atpE2 has potential applications in synthetic biology for ATP generation systems. Researchers can develop minimal ATP-producing systems by following these approaches:

• Minimal reconstituted systems:

  • Design simplified ATP synthase complexes using atpE2 as part of engineered c-rings

  • Reconstitute with essential components (subunit a, F1 sector) in liposomes

  • Engineer proton gradient generation through light-driven pumps or chemical gradients

• Integration with alternative energy sources:

  • Couple to hydrogen oxidation systems (hydrogenases)

  • Recent work has demonstrated that hydrogenase-driven ATP synthesis is possible using atmospheric hydrogen, where a minimal respiratory chain containing ATP synthase can generate up to two ATP molecules per H2 oxidized

  • Similar principles could be applied using atpE2-containing ATP synthases

• Potential productivity metrics:

  System	ATP production rate	Energy source	Reference
  Hydrogenase-coupled	~2 ATP/H2	Atmospheric H2
  Light-driven	~3 ATP/photon	Light	Theoretical
  Chemical gradient	Variable	pH differential	Estimated

• Optimization strategies:

  • Engineer c-ring size to adjust H+/ATP ratio

  • Modify proton binding sites for altered specificity or kinetics

  • Develop hybrid systems with components from different organisms

These applications represent a frontier in synthetic biology, where engineered ATP production systems could power artificial cells or provide energy for biotechnological processes .

What is the role of atpE2 in the bioenergetic adaptations of Pelobacter propionicus to its environmental niche?

Pelobacter propionicus inhabits anaerobic environments and has evolved specialized metabolic and bioenergetic adaptations. The atpE2 protein likely plays a key role in these adaptations:

• Environmental adaptation:

  • Anaerobic lifestyle requires efficient energy conservation

  • Limited energy sources in anaerobic environments necessitate optimized ATP synthase function

  • The c-ring structure may be adapted for optimal function at lower proton motive force

• Comparative analysis with related species:

  • The c-subunit composition may reflect adaptation to specific environmental conditions

  • The size of the c-ring (number of c-subunits) determines the H+/ATP ratio, thus affecting the ATP synthesis efficiency

• Research approaches to study environmental adaptations:

  • Functional characterization at different pH values and ion concentrations

  • Comparison of ATP synthesis rates under conditions mimicking natural habitat

  • Mutagenesis studies to identify residues important for environmental adaptation

  • Genomic analysis to identify regulatory elements controlling atpE2 expression

• Evolutionary significance:

  • Comparison with c-subunits from phylogenetically related organisms

  • Identification of conserved vs. variable regions that may reflect adaptation

Understanding the specific adaptations of atpE2 provides insights into how energy conservation mechanisms evolve to support life in extreme or nutrient-limited environments. This knowledge could inform the development of bioenergetic systems for biotechnological applications in similar environments.

What are the common challenges in working with recombinant atpE2 and how can they be addressed?

Working with membrane proteins like atpE2 presents several technical challenges that researchers should anticipate:

• Expression challenges:

  • Problem: Low expression yields

  • Solution: Test multiple E. coli strains (C41/C43 designed for membrane proteins); optimize induction conditions; consider fusion partners (SUMO, MBP); use specialized media formulations

  • Problem: Formation of inclusion bodies

  • Solution: Lower induction temperature (16-18°C); reduce inducer concentration; co-express with chaperones

• Purification difficulties:

  • Problem: Poor solubilization

  • Solution: Screen multiple detergents (DDM, LMNG, UDM); optimize detergent:protein ratio; consider milder extraction conditions

  • Problem: Protein aggregation during purification

  • Solution: Maintain detergent above CMC in all buffers; add glycerol (10%) or specific lipids; avoid freeze-thaw cycles

• Reconstitution issues:

  • Problem: Low incorporation efficiency into liposomes

  • Solution: Optimize protein:lipid ratio; test different reconstitution methods (detergent removal rates); verify incorporation by density gradient centrifugation

  • Problem: Random orientation in liposomes

  • Solution: Use pH gradient during reconstitution; engineer orientation-specific tags

• Storage considerations:

  • Recommended storage: -80°C in buffer containing 6% trehalose, pH 8.0

  • Avoid repeated freeze-thaw cycles

  • For working stocks, store at 4°C for up to one week

  • After reconstitution, use concentration range of 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage

How can researchers verify the functional integrity of purified recombinant atpE2?

Verifying the functional integrity of purified atpE2 is crucial before proceeding with experimental applications. Several complementary approaches can be used:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy: Confirms secondary structure content

  • Fluorescence spectroscopy: Monitors tertiary structure through intrinsic tryptophan fluorescence

  • Size-exclusion chromatography: Ensures monodispersity and proper oligomeric state

• Binding assays:

  • Detergent/lipid binding: Isothermal titration calorimetry (ITC) to measure binding of specific lipids

  • Inhibitor binding: Measuring binding of known ATP synthase inhibitors (oligomycin, DCCD)

• Functional reconstitution tests:

  • Proton translocation: Fluorescence-based assays using pH-sensitive dyes

  • ATP synthesis/hydrolysis: When reconstituted with other ATP synthase components

• Molecular probes:

  • Site-specific labeling: Introducing fluorescent or spin labels at specific positions

  • Accessibility assays: Chemical modification of key residues (e.g., conserved glutamate)

• Quality control metrics:

  Test	Expected outcome	Troubleshooting
  SDS-PAGE	Single band at ~10 kDa	Re-purify if multiple bands present
  SEC-MALS	Appropriate oligomeric state	Optimize detergent/buffer conditions
  CD spectroscopy	High α-helical content	Adjust purification protocol if structure compromised
  Proton translocation	pH-dependent response	Check reconstitution efficiency