Recombinant Azoarcus sp. ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c (atpE) in Azoarcus sp.?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of ATP synthase in Azoarcus sp. While the search results do not provide specific information about atpE, we can extrapolate from related ATP synthase subunits in Azoarcus. The protein likely forms part of the membrane-embedded proton channel that facilitates proton translocation across the membrane. This proton movement drives the rotational mechanism that powers ATP synthesis.

Based on related ATP synthase subunits in Azoarcus sp., the atpE protein is likely a small, hydrophobic protein with multiple transmembrane segments. For comparison, ATP synthase subunit a (atpB) in Azoarcus sp. is 282 amino acids in length and contains several transmembrane regions as evident from its amino acid sequence . The high hydrophobicity is necessary for embedding within the membrane bilayer where it functions as part of the proton channel.

ATP synthase plays an important role in human wellbeing, with its dysfunction being associated with a wide range of illnesses including tuberculosis, neuropathy, Alzheimer's, and Parkinson's . In bacterial systems like Azoarcus, ATP synthase is especially crucial during energy-limited states.

How does Azoarcus sp. ATP synthase subunit c differ from other ATP synthase subunits?

ATP synthase comprises multiple subunits that work together in a complex. In Azoarcus sp., several ATP synthase subunits have been characterized:

Subunit c (atpE) differs from the others primarily in its arrangement as a ring structure within the F0 sector. While subunit a (atpB) forms part of the static proton channel, subunit c typically forms a ring of multiple copies that rotates during proton translocation. Subunit b (atpF) serves as a stator connecting the F0 and F1 sectors .

The amino acid sequence of ATP synthase subunit a in Azoarcus reveals hydrophobic regions consistent with its transmembrane nature: "MATEHAPTASEYVVHHLTHLNSTGHAQTSIVDFSVINVDSMFYSVLLGLLTVFLLWLAAR KATAGVPGRFQGFVELLVEMVADQAKGIIHSAESRKFVAPLALTVFVWIFLMNAMDMLPV DLLPRIWEGVYASAGGDPHHAYMRVVPTADLSATLGMSCGVLLLCLYYNVKIKGVSGWVH ELFTAPFGSHPLLYPINFAMQIIEFVAKTVSHGMRLFGNMYAGELIFILIALLGSTATVF GFVGHIVAGSIWAIFHILIITLQAFIFMMLTLVYIGQAHEGH" . Subunit c would likely display similar hydrophobic character but with a smaller size and simpler structure.

What are the optimal expression systems for recombinant Azoarcus sp. ATP synthase subunit c?

When expressing recombinant ATP synthase subunits from Azoarcus sp., E. coli is commonly used as the expression host. Based on the expression systems used for other Azoarcus ATP synthase subunits, the following considerations are important:

• Expression host selection: E. coli is the preferred expression system for Azoarcus sp. ATP synthase subunits, as demonstrated with the successful expression of ATP synthase subunit a (atpB) and other subunits .

• Vector and tag selection: N-terminal His-tagging has proven effective for ATP synthase subunit a, facilitating purification while maintaining protein structure and function . For membrane proteins like subunit c, this approach would likely be suitable as well.

• Expression conditions: Optimal conditions typically include growth at lower temperatures (25-30°C) after induction to allow proper folding of membrane proteins, with expression periods of 4-16 hours depending on the construct.

It's worth noting that expression of membrane proteins like ATP synthase subunit c can be challenging due to their hydrophobic nature. Alternative expression systems including yeast, baculovirus, or mammalian cell systems may be considered for difficult-to-express constructs, as mentioned in the available options for other Azoarcus sp. proteins .

What purification strategies are most effective for recombinant Azoarcus sp. ATP synthase subunit c?

Purification of recombinant ATP synthase subunit c requires careful consideration of its membrane protein nature. Based on protocols used for similar ATP synthase subunits, the following purification strategy would be effective:

• Initial preparation: Following expression, centrifuge the bacterial culture and resuspend the cell pellet in an appropriate buffer. For ATP synthase subunit a from Azoarcus sp., a Tris/PBS-based buffer with 6% Trehalose at pH 8.0 has been used successfully .

• Cell lysis: Use sonication or pressure-based disruption methods in the presence of appropriate detergents to solubilize membrane proteins.

• Affinity chromatography: For His-tagged constructs, use immobilized metal affinity chromatography (IMAC) with Ni-NTA resin. Wash with increasing concentrations of imidazole to remove non-specifically bound proteins before elution with high imidazole.

• Quality control: SDS-PAGE analysis can confirm protein purity, which should exceed 90% for research applications, as specified for ATP synthase subunit a .

• Storage considerations: Lyophilization is an effective preservation method. Store the lyophilized protein at -20°C to -80°C. Reconstitution should be done in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with addition of 5-50% glycerol (typically 50%) for long-term storage. Working aliquots can be stored at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided .

How can researchers verify the functional integrity of purified Azoarcus sp. ATP synthase subunit c?

Verification of functional integrity is crucial for research applications. For ATP synthase subunit c, the following methods can be employed:

• Structural analysis:

  • Circular dichroism (CD) spectroscopy to verify secondary structure elements

  • Size exclusion chromatography to confirm proper oligomeric state

  • Mass spectrometry to verify the exact mass and potential post-translational modifications

• Functional assays:

  • Reconstitution into liposomes for proton translocation assays

  • Assembly with other ATP synthase subunits to form functional complexes

  • ATP hydrolysis/synthesis assays with reconstituted complexes

• Binding studies:

  • Interaction analysis with known ATP synthase inhibitors

  • Co-immunoprecipitation with other ATP synthase subunits

These verification methods are essential since ATP synthase functionality depends on proper folding and assembly of all subunits, including subunit c, which forms the critical c-ring within the F0 sector.

What are the recommended protocols for reconstituting Azoarcus sp. ATP synthase subunit c into liposomes?

Reconstitution of ATP synthase subunit c into liposomes allows for functional studies of proton translocation. The following protocol outline is recommended:

• Liposome preparation:

  • Prepare a lipid mixture containing E. coli polar lipids and phosphatidylcholine (3:1 ratio)

  • Dry lipids under nitrogen and resuspend in reconstitution buffer

  • Subject to freeze-thaw cycles and extrusion through polycarbonate filters

• Protein incorporation:

  • Add detergent-solubilized purified ATP synthase subunit c to preformed liposomes

  • Gradually remove detergent using Bio-Beads or dialysis

  • For complete ATP synthase complex reconstitution, combine with other purified subunits

• Functional verification:

  • Measure proton pumping using pH-sensitive fluorescent dyes

  • Assess ATP synthesis capability if reconstituted with complete complex

For accurate assessment of subunit c function, comparison with known ATP synthase inhibitors can provide valuable controls. ATP synthase is targeted by several compounds, including those under development for treatment of bacterial infections like tuberculosis .

How can site-directed mutagenesis be employed to study critical residues in Azoarcus sp. ATP synthase subunit c?

Site-directed mutagenesis is a powerful approach for investigating the function of specific amino acid residues in ATP synthase subunit c. The following protocol outline can guide such studies:

• Target residue identification:

  • Select conserved residues based on sequence alignment with other bacterial species

  • Focus on residues in the proton-binding site (typically including a conserved carboxylic acid residue)

  • Consider residues at the interface with other subunits

• Mutagenesis strategy:

  • Design mutagenic primers for QuikChange or overlap extension PCR

  • Introduce conservative and non-conservative substitutions

  • Verify mutations by sequencing

• Functional characterization:

  • Express and purify mutant proteins using the same methods as wild-type

  • Compare structural properties (CD spectroscopy, thermal stability)

  • Assess proton translocation efficiency in reconstituted systems

  • Measure impacts on ATP synthesis in complete complexes

• Data analysis:

  • Quantify changes in enzyme kinetics (Km, Vmax)

  • Correlate structural changes with functional outcomes

This approach has been valuable in identifying critical residues in ATP synthase from other organisms and could reveal important insights into the specific properties of Azoarcus sp. ATP synthase subunit c.

What approaches can be used to identify potential inhibitors of Azoarcus sp. ATP synthase subunit c?

ATP synthase inhibitors have significant research and therapeutic potential. For identifying inhibitors of Azoarcus sp. ATP synthase subunit c, the following methodologies are recommended:

• High-throughput screening approaches:

  • ATP synthesis inhibition assays using reconstituted systems

  • Fluorescence-based proton translocation assays

  • Thermal shift assays to identify compounds that stabilize the protein

• Structure-based drug design:

  • Homology modeling based on known ATP synthase structures

  • Molecular docking of compound libraries

  • Molecular dynamics simulations of protein-inhibitor interactions

• Validation methods:

  • Isothermal titration calorimetry (ITC) for binding affinity determination

  • Surface plasmon resonance (SPR) for binding kinetics

  • X-ray crystallography or cryo-EM of inhibitor-bound structures

ATP synthase subunit c has been identified as a promising drug target for treating various bacterial infections. Studies have shown that targeting ATP synthase inhibits biofilm formation and acid production in bacteria like Streptococcus mutans, and it serves as an alternative target in Mycobacterium tuberculosis when resistance to other drugs emerges .

How can Azoarcus sp. ATP synthase subunit c be used to study bacterial energy metabolism under different environmental conditions?

ATP synthase plays a crucial role in bacterial adaptation to changing environments. Using recombinant Azoarcus sp. ATP synthase subunit c, researchers can investigate:

• Response to environmental stressors:

  • Expression levels under different pH, temperature, or oxygen conditions

  • Post-translational modifications in response to stress

  • Structural changes affecting function

• Methodological approach:

  • Quantitative PCR to measure gene expression

  • Western blotting to assess protein levels

  • Blue native PAGE to examine complex assembly

  • Enzyme activity assays under varying conditions

• Experimental design:

  • Grow Azoarcus cultures under controlled environmental conditions

  • Isolate native ATP synthase complexes

  • Compare with reconstituted systems containing recombinant subunit c

  • Measure ATP synthesis rates and proton translocation efficiency

The data from such studies can be presented in comparative tables showing ATP synthase activity under different conditions:

What are the comparative differences between ATP synthase subunit c from Azoarcus sp. and clinically relevant bacterial pathogens?

Understanding the differences between ATP synthase subunit c from various bacterial species can inform both basic research and drug development efforts:

• Sequence and structural comparison:

  • Perform multiple sequence alignments of subunit c from Azoarcus and pathogenic bacteria

  • Identify conserved and variable regions

  • Model structural differences that might affect inhibitor binding

• Functional differences:

  • Compare proton binding affinities

  • Assess ATP synthesis rates

  • Evaluate responses to known inhibitors

• Research applications:

  • Develop selective inhibitors targeting pathogen-specific features

  • Use Azoarcus as a non-pathogenic model system for studying ATP synthase function

  • Design chimeric proteins to investigate structure-function relationships

ATP synthase has been associated with various human diseases and serves as a drug target in treating infections, including those caused by Mycobacterium tuberculosis and Streptococcus mutans . Comparative studies can reveal why certain inhibitors are effective against specific bacterial species while sparing others.