Recombinant Mycoplasma gallisepticum ATP synthase subunit b (atpF)

What is the basic structure of Mycoplasma gallisepticum ATP synthase and the role of subunit b?

ATP synthase in M. gallisepticum, like in other bacteria, is a multi-subunit enzyme complex composed of the F₁ catalytic domain and the F₀ membrane domain. The subunit b (atpF) serves as part of the peripheral stalk connecting F₁ and F₀ domains, thereby playing a crucial role in maintaining structural integrity during the rotational catalysis of ATP synthesis. Unlike conventional bacteria, M. gallisepticum has a reduced genome and lacks a cell wall, which affects membrane protein organization and potentially the ATP synthase complex structure . The atpF gene in M. gallisepticum has been sequenced and characterized as part of the atp operon, revealing significant homology with other bacterial ATP synthases but with distinct adaptations to the mycoplasma environment .

Research methodological consideration: When investigating atpF structure, researchers should account for the unique membrane composition of M. gallisepticum, which lacks a cell wall and contains sterols - unusual for prokaryotes but characteristic of mycoplasmas.

How does the ATP synthase complex function in M. gallisepticum compared to other bacteria?

Methodologically, researchers investigating ATP synthase function should implement membrane potential measurements specific to mycoplasma cells, as their unique membrane composition can affect proton gradient maintenance differently than in model organisms like E. coli.

What are the optimal expression systems for producing recombinant M. gallisepticum atpF?

The expression of recombinant M. gallisepticum atpF presents several challenges due to its hydrophobic domains and potential toxicity. Based on comparative research with similar membrane proteins, the following expression systems provide optimal results:

For methodological implementation, researchers should consider codon optimization for E. coli expression, as M. gallisepticum has a different codon usage pattern. The CAI (Codon Adaptation Index) should be optimized to approximately 0.97, and the GC content adjusted to around 51.7% for optimal expression in E. coli systems .

What purification strategies effectively isolate recombinant atpF while maintaining protein stability?

Purification of recombinant atpF requires specific approaches to maintain protein stability:

• Initial extraction using mild detergents (n-dodecyl-β-D-maltoside at 1-2% concentration) preserves native-like conformation

• Immobilized metal affinity chromatography (IMAC) with histidine tags positioned at the C-terminus rather than N-terminus to avoid interference with membrane integration

• Size exclusion chromatography in the presence of stabilizing agents (glycerol 10% v/v)

Methodologically, researchers should monitor protein stability throughout purification using circular dichroism spectroscopy to ensure secondary structure maintenance, particularly the alpha-helical content which is critical for atpF function.

What are the most effective crystallization approaches for structural determination of M. gallisepticum atpF?

Crystallization of membrane proteins like atpF remains challenging. Based on successful approaches with similar ATP synthase components, researchers should consider:

• Lipidic cubic phase (LCP) crystallization, which better mimics the membrane environment

• Detergent screening focusing on maltosides and glucosides at concentrations just above their critical micelle concentration

• Co-crystallization with antibody fragments to increase polar surface area

Methodological consideration: Successful crystallization often requires protein engineering to remove flexible regions while maintaining functional domains. For atpF, computational analysis predicts that removing 5-7 amino acids from the C-terminus may improve crystallization prospects without compromising structural integrity.

How can cryo-EM be optimized for structural analysis of recombinant atpF in the context of the ATP synthase complex?

Cryo-EM represents a powerful alternative to crystallography for membrane protein structural analysis. For atpF research:

• Sample preparation should include mild cross-linking (0.1% glutaraldehyde for 5 minutes) to stabilize atpF associations within the ATP synthase complex

• Vitrification conditions should be optimized with grid types containing thin carbon support films

• Image processing should implement focused refinement techniques to enhance resolution in the peripheral stalk region containing atpF

Methodologically, researchers should compare structures obtained with and without lipid nanodiscs to evaluate potential conformational differences in different membrane-mimetic environments.

What assays can effectively measure the contribution of atpF to ATP synthase activity in recombinant systems?

Functional characterization of atpF requires specialized assays that distinguish its structural contribution from catalytic activities:

• Reconstitution assays incorporating purified recombinant atpF into liposomes with other ATP synthase subunits to measure restoration of ATP synthesis

• FRET-based interaction assays to quantify binding affinities between atpF and partner subunits

• Cross-linking studies followed by mass spectrometry to map interaction interfaces

For methodological implementation, researchers should establish a complementation system using atpF-deficient bacterial strains to quantitatively assess function restoration, similar to approaches used with B. subtilis ATP synthase mutants .

How does site-directed mutagenesis of conserved residues impact atpF function within the ATP synthase complex?

Site-directed mutagenesis provides valuable insights into structure-function relationships. Key experimental approaches include:

Methodologically, researchers should implement complementary biophysical techniques (differential scanning calorimetry, isothermal titration calorimetry) to quantify how mutations affect thermodynamic stability and interaction energetics.

How can recombinant atpF be incorporated into multi-epitope vaccine strategies against M. gallisepticum?

Recombinant atpF shows potential as a vaccine component due to its surface exposure and conservation. Research approaches include:

• Epitope mapping to identify immunogenic regions specific to M. gallisepticum atpF using computational tools

• Construction of chimeric proteins incorporating atpF epitopes with appropriate adjuvants

• Testing various delivery systems including nanoparticles and viral vectors

Based on computational vaccinology approaches similar to those used for other M. gallisepticum proteins, the inclusion of atpF epitopes can enhance vaccine efficacy by broadening the immune response . Methodologically, researchers should implement both B-cell and T-cell epitope prediction algorithms, with priority given to epitopes that score >0.6 on antigenicity scales and demonstrate species specificity.

What is the immunogenic profile of recombinant atpF and how does it compare to other M. gallisepticum antigens?

The immunogenic profile of atpF involves:

• Predominantly T-helper cell epitopes located in the cytoplasmic domains

• Limited B-cell epitopes restricted to exposed loops

• Significant conservation across M. gallisepticum strains, suggesting value as a broad-spectrum antigen

How can CRISPR-Cas systems be optimized for studying atpF function in M. gallisepticum?

CRISPR-Cas gene editing presents unique challenges in mycoplasmas due to their AT-rich genomes and lack of non-homologous end joining repair pathways. For atpF research, optimized approaches include:

• Design of sgRNAs with minimal off-target potential accounting for the AT-rich genome

• Use of Cas9 nickase rather than nuclease to reduce toxicity

• Implementation of donor templates with extended homology arms (>1kb)

Methodologically, researchers should establish stably transformed CRISPR systems rather than transient expression, using tetracycline-inducible promoters to control Cas9 expression levels in M. gallisepticum.

What are the implications of atpF structural variations across different M. gallisepticum strains for ATP synthase function?

Strain variation analysis of atpF sequences reveals:

• Conservation of transmembrane domains across strains

• Variable regions concentrated in cytoplasmic domains

• Correlation between specific polymorphisms and virulence phenotypes

This suggests potential adaptation of ATP synthase function to different host environments and virulence states. For methodological implementation, researchers should conduct comparative biochemical analysis of ATP synthase activity across recombinant atpF variants representing different strains, using proteoliposome reconstitution systems to quantify functional differences.

How can researchers address solubility issues when working with recombinant atpF?

Solubility challenges with atpF stem from its hydrophobic character. Effective strategies include:

• Expression as fusion proteins with solubility enhancers (MBP, SUMO) positioned at the N-terminus

• Systematic detergent screening focusing on mild non-ionic detergents

• Use of amphipols or nanodiscs for long-term stability

Based on similar membrane proteins, biochemical analysis indicates that fusion tags increase solubility by 60-70%, while proper detergent selection can improve homogeneity by 40-50% . Methodologically, researchers should implement small-scale parallel screening approaches before scaling up, using dynamic light scattering to monitor aggregation states.

What strategies can overcome the challenges of antibody production against conserved atpF epitopes?

Generating specific antibodies against atpF presents difficulties due to high conservation across bacterial species and potential immunodominance of certain epitopes. Effective approaches include:

• Use of synthetic peptides corresponding to M. gallisepticum-specific regions rather than full-length protein

• Immunization protocols with DNA prime-protein boost strategies

• Screening of monoclonal antibody libraries against structural models

For methodological implementation, researchers should employ epitope mapping techniques to confirm antibody specificity, including competitive ELISA and peptide arrays, ensuring reagents specifically recognize M. gallisepticum atpF rather than homologs from other species.