Recombinant Alteromonas macleodii ATP synthase subunit alpha (atpA), partial

What is ATP synthase α subunit and what is its role in cellular energy production?

ATP synthase α subunit is one of the key components of the F1 domain (the catalytic head) of ATP synthase, the enzyme complex that functions as the "turbine" of cellular power plants. This enzyme synthesizes adenosine triphosphate (ATP) by utilizing the energy stored in proton gradients across membranes. The α subunit contains nucleotide-binding domains that interact with other subunits to drive conformational changes essential for catalysis .

What functional domains are present in ATP synthase α subunits?

ATP synthase α subunits typically contain several functional domains:

Research has shown that in some organisms, specific regions of the α subunit have evolved additional functions. For example, in zebrafish, the N-terminal 65 residues of ATP5A1 are critical for its antibacterial activity against both gram-positive and gram-negative bacteria .

What expression systems are optimal for producing recombinant A. macleodii ATP synthase α subunit?

Based on commercial product information, A. macleodii ATP synthase α subunit is successfully produced using baculovirus expression systems . This system, which uses insect cells infected with recombinant baculovirus containing the target gene, is advantageous for:

• Producing proteins that require specific post-translational modifications

• Expressing proteins that may be toxic to bacterial hosts

• Achieving higher yields of properly folded protein compared to some bacterial systems

• Producing proteins that form complexes or require chaperones for proper folding

For laboratory-scale production, the following methodological approach is recommended:

• Clone the atpA gene from A. macleodii genomic DNA using PCR

• Insert the gene into a suitable baculovirus transfer vector

• Generate recombinant baculovirus in insect cells

• Optimize expression conditions (time, temperature, MOI)

• Purify using affinity chromatography based on the tag selected during cloning

What are the optimal storage and handling conditions for recombinant ATP synthase α subunit?

According to product documentation, the following storage and handling conditions are recommended for maintaining protein stability and activity :

It is advisable to centrifuge the vial briefly before opening to bring contents to the bottom. For applications requiring buffer exchange, consider dialysis or size exclusion chromatography to maintain protein stability .

What methods are used to assess the enzymatic activity of recombinant ATP synthase α subunit?

Several methodological approaches can be employed to characterize the enzymatic activity of recombinant ATP synthase α subunit:

• ATP synthesis/hydrolysis assays: These can be performed on the isolated α subunit or, more effectively, on reconstituted complexes containing multiple ATP synthase subunits. ATP hydrolysis can be measured by quantifying released inorganic phosphate using colorimetric methods.

• Single-molecule rotation assays: As demonstrated in studies with mycobacterial ATP synthase, single-molecule assays can provide insights into how specific domains affect the angular velocity of the power-stroke after ATP binding .

• Binding studies: Isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) can be used to characterize nucleotide binding properties.

• Structural studies: Solution X-ray scattering and NMR can reveal structural features and conformational changes, as demonstrated in studies of mycobacterial ATP synthase .

Beyond energy production, what alternative functions have been identified for ATP synthase α subunits?

Research has revealed surprising moonlighting functions for ATP synthase α subunits beyond their canonical role in energy production:

• Antimicrobial activity: In zebrafish, ATP5A1 (ATP synthase α subunit) functions as a pattern recognition receptor capable of binding lipoteichoic acid (LTA) and lipopolysaccharide (LPS). It can directly inhibit the growth of both gram-positive and gram-negative bacteria by disrupting bacterial membranes through a combined action of membrane depolarization and permeabilization .

• Pathogen resistance in embryonic development: Microinjection of recombinant ATP5A1 into early zebrafish embryos promotes resistance against pathogenic Aeromonas hydrophila challenge. This protective effect is specifically attributable to the N-terminal 65 residues of ATP5A1 .

These findings suggest that ATP synthase α subunits from various species may have evolved additional functions that could be relevant in different biological contexts.

How can site-directed mutagenesis be used to study structure-function relationships in A. macleodii ATP synthase α subunit?

Site-directed mutagenesis offers a powerful approach to dissect the functional domains of ATP synthase α subunit:

• Nucleotide-binding sites: Mutations in residues involved in ATP/ADP binding can help understand the catalytic mechanism. Key residues can be identified through sequence alignment with well-characterized homologs.

• Subunit interfaces: Mutating residues at interfaces with other subunits (particularly β and γ) can reveal how subunit interactions contribute to the rotary mechanism of ATP synthase.

• Potential antimicrobial domains: Based on findings in zebrafish ATP5A1, where the N-terminal 65 residues confer antibacterial activity, similar regions in A. macleodii ATP synthase α could be explored through deletion mutants or chimeric constructs .

The methodological approach should include:

• Expression of mutant proteins using the same system as the wild-type

• Comparative biophysical characterization (stability, folding)

• Functional assays relevant to the domain being studied

• Structural analysis when possible

How does petrobactin production by A. macleodii relate to ATP synthase function?

A. macleodii produces petrobactin, a siderophore that increases the bioavailability of specific iron sources . While direct connections between petrobactin production and ATP synthase function are not explicitly established in the literature, several potential relationships can be hypothesized:

• Energy requirements: Siderophore biosynthesis requires ATP. The efficiency of ATP synthase directly impacts the cell's energy budget and consequently its capacity to produce petrobactin.

• Iron-sulfur clusters: Some ATP synthase subunits contain iron-sulfur clusters essential for electron transfer. Iron acquisition through petrobactin could indirectly support ATP synthase assembly and function.

• Regulatory connections: Iron availability, mediated by petrobactin, might regulate ATP synthase expression or activity through global regulatory networks responding to iron status.

Experimental approaches to investigate these relationships could include:

• Comparative proteomics and transcriptomics under iron-limited conditions

• ATP synthase activity assays in petrobactin-deficient mutants (e.g., ΔasbB::kmr)

• Measurement of intracellular ATP levels in relation to petrobactin production

What techniques are used to study the assembly of ATP synthase complexes?

Understanding ATP synthase assembly is crucial for comprehending its function. Recent research has revealed that molecular chaperones, particularly Hsp70, play essential roles in ATP synthase assembly . The following methodologies can be employed:

• Protein-protein interaction studies: Techniques such as co-immunoprecipitation, yeast two-hybrid, or proximity labeling can identify interaction partners during assembly.

• Time-resolved proteomics: Pulse-chase experiments combined with mass spectrometry can track the assembly process temporally.

• Cryo-electron microscopy: This technique can visualize assembly intermediates and resolve structural details of partially assembled complexes.

• Genetic approaches: As demonstrated in studies with Hsp70, researchers discovered that this chaperone promotes the assembly of ATP synthase by being involved with partner proteins in the assembly of the catalytic head and monitoring the linkage of the catalytic head to the stator .

What are common challenges in preserving activity of recombinant ATP synthase α subunit?

Several challenges may arise when working with recombinant ATP synthase α subunit:

How can researchers verify the authenticity and integrity of recombinant A. macleodii ATP synthase α subunit?

Verifying protein authenticity and integrity is crucial for reliable research. The following methods are recommended:

• SDS-PAGE and Western blotting: Confirm the correct molecular weight and immunoreactivity with specific antibodies.

• Mass spectrometry: Peptide mass fingerprinting can confirm the protein identity and detect post-translational modifications or truncations.

• Circular dichroism: Assess secondary structure content to ensure proper folding.

• Activity assays: Functional tests specific to ATP synthase α subunit can confirm biological activity.

• Thermal shift assays: These can provide information about protein stability and proper folding.

What controls should be included when studying potential antimicrobial properties of ATP synthase α subunits?

When investigating potential antimicrobial properties similar to those observed in zebrafish ATP5A1 , the following controls should be included:

• Negative controls: Include buffer-only conditions and irrelevant proteins of similar size/charge.

• Domain-specific controls: Test isolated domains (e.g., N-terminal fragments, C-terminal fragments) to identify the active region.

• Heat-inactivated controls: Compare activity with heat-denatured protein to distinguish between specific activity and non-specific effects.

• Specificity controls: Test activity against multiple bacterial species and eukaryotic cells to assess specificity.

• Mechanistic controls: Include membrane potential indicators and permeability assays to verify the proposed mechanism of action.