Recombinant Mycoplasma mycoides subsp. mycoides SC ATP synthase subunit c (atpE)

What is the function of ATP synthase subunit c (atpE) in Mycoplasma mycoides subsp. mycoides SC?

ATP synthase subunit c (atpE) is a critical component of the F-type ATP synthase complex in Mycoplasma mycoides subsp. mycoides SC (Mmm SC). This protein functions as part of the membrane-embedded F0 sector of ATP synthase, forming an oligomeric ring structure that facilitates proton translocation across the membrane. This proton flow drives the conformational changes in the F1 sector that enable ATP synthesis. In Mycoplasma species, which lack a cell wall and have simplified metabolic pathways, the ATP synthase complex is particularly crucial for energy metabolism and survival .

How does the atpE protein from Mycoplasma mycoides compare to homologous proteins in other Mycoplasma species?

The atpE protein from Mycoplasma mycoides subsp. mycoides SC shares structural and functional similarities with homologous proteins in other Mycoplasma species, though with some notable differences:

• Sequence analysis reveals that Mmm SC atpE protein (101 amino acids) has conserved functional domains typical of ATP synthase c subunits, including the transmembrane helices and the ion-binding site .

• Compared to Mycoplasma pneumoniae atpE, the Mmm SC version shows similar functional properties but distinct immunogenic epitopes, which is relevant for diagnostic applications .

• Unlike some other bacterial species, Mycoplasma atpE proteins are relatively small and lack certain regulatory domains found in more complex organisms, reflecting the minimal genome strategy of Mycoplasma species .

Comparative proteomic analyses between Mycoplasma species have shown that ATP synthase components, including atpE, are among the conserved secretory proteins, suggesting their potential roles beyond energy metabolism, possibly in virulence or host interactions .

What are the optimal expression systems for producing recombinant Mycoplasma mycoides atpE protein?

Based on current research protocols, the optimal expression systems for recombinant Mmm SC atpE include:

• E. coli expression system: Most commonly used due to:

  • High yield (typically 2-5 mg/L of culture)

  • Compatibility with N-terminal His-tagging for purification

  • Successful expression of the full-length protein (1-101aa)

• Yeast expression system: Alternative option when:

  • Post-translational modifications are desired

  • Protein folding issues occur in E. coli

  • Higher purity (>85% by SDS-PAGE) is required

Methodology considerations:

• For E. coli expression, BL21(DE3) strains with T7 promoter systems yield better results than other strains

• Induction at lower temperatures (16-18°C) improves solubility

• Inclusion of 0.5% glucose in the pre-induction medium helps reduce basal expression

• IPTG concentrations between 0.1-0.5 mM provide optimal induction without toxicity

What purification strategies yield the highest purity recombinant atpE protein for structural studies?

For high-purity recombinant atpE protein suitable for structural studies, a multi-step purification strategy is recommended:

• Initial capture using affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged proteins

  • Binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Elution with imidazole gradient (20-250 mM)

• Secondary purification steps:

  • Size exclusion chromatography using Superdex 75 column

  • Ion exchange chromatography (IEX) using Q-Sepharose column

• Detergent considerations for membrane protein isolation:

  • Mild detergents like n-dodecyl-β-D-maltoside (DDM) or n-octyl-β-D-glucopyranoside (OG) at 0.05-0.1% maintain protein stability

  • Detergent exchange during purification improves final purity

This approach consistently yields >95% pure protein suitable for crystallography or cryo-EM studies, with typical final yields of 1-2 mg per liter of initial culture .

How can researchers distinguish between different ATP synthase subunits in Mycoplasma species using analytical methods?

Distinguishing between different ATP synthase subunits requires a combination of analytical approaches:

• Mass spectrometry-based differentiation:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can identify unique peptide signatures

  • The atpE subunit produces characteristic peptide fragments after trypsin digestion

  • Comparative analysis with atpA (alpha) and atpD (beta) subunits shows distinct peptide mass fingerprints

• Immunological differentiation:

  • Subunit-specific antibodies can be developed targeting unique epitopes

  • Western blotting with anti-His antibodies can identify tagged recombinant subunits

  • For atpE, antibodies against the C-terminal region show minimal cross-reactivity with other subunits

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy reveals different secondary structure compositions

  • Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) distinguishes oligomeric states

  • AtpE typically shows characteristic alpha-helical signatures in CD analysis

• Sequence-based identification:

  • PCR amplification using subunit-specific primers

  • DNA sequencing of the amplified regions

  • AtpE can be distinguished from atpA and atpD based on size (303 bp for atpE versus 1542 bp for atpA)

What experimental approaches can determine the oligomeric state of atpE in native Mycoplasma membranes?

Determining the native oligomeric state of atpE requires specialized membrane protein analysis techniques:

• Crosslinking studies:

  • Chemical crosslinkers like disuccinimidyl suberate (DSS) or glutaraldehyde can capture native oligomeric states

  • Analysis by SDS-PAGE reveals the molecular weight of crosslinked complexes

  • Western blotting with anti-atpE antibodies confirms subunit identity

• Blue native PAGE analysis:

  • Gentle solubilization using digitonin (0.5-1%) preserves native complexes

  • BN-PAGE separates intact membrane protein complexes

  • Second dimension SDS-PAGE identifies constituent subunits

• Cryo-electron microscopy of native membranes:

  • Direct visualization of ATP synthase complexes in native lipid environment

  • Single-particle analysis determines the number of c-subunits in the c-ring

  • In Mycoplasma species, the c-ring typically contains 8-10 atpE subunits

• Mass photometry:

  • Label-free technique for determining molecular mass distribution of membrane protein complexes

  • Can distinguish between monomeric atpE (~10 kDa) and oligomeric c-rings (~80-100 kDa)

  • Requires minimal sample amounts (nanograms)

These approaches collectively provide complementary evidence for the native oligomeric state of atpE in Mycoplasma membranes .

How can recombinant atpE protein be used to develop serological assays for Mycoplasma mycoides detection?

Recombinant atpE protein serves as a valuable antigen for developing serological assays for Mycoplasma mycoides detection through these methodological approaches:

• ELISA development:

  • Recombinant atpE can be immobilized on microtiter plates at 1-5 μg/ml in carbonate buffer (pH 9.6)

  • Blocking with 5% skim milk reduces non-specific binding

  • Serum samples are typically diluted 1:100 to 1:500

  • Detection using species-appropriate HRP-conjugated secondary antibodies

  • Optimization of cutoff values using ROC curve analysis with known positive and negative samples

• Immunoblot assays:

  • Western blot using purified recombinant atpE (0.5-1 μg per lane)

  • Transfer to PVDF membranes at 25V for 30 minutes shows optimal protein retention

  • Diluted serum samples (1:200) are incubated overnight at 4°C

  • Visual or densitometric analysis of band intensity correlates with antibody levels

• Multiplex serological assays:

  • Coupling atpE to differentially colored microspheres allows multiplexing with other antigens

  • Combination with other Mycoplasma antigens (like atpA or adhesins) increases diagnostic sensitivity

  • Analysis using flow cytometry-based platforms enhances quantitative assessment

Research shows that atpE-based assays achieve 85-92% sensitivity and 89-95% specificity for Mycoplasma mycoides detection, making them valuable diagnostic tools, particularly when combined with other antigenic markers .

What are the immunogenic epitopes in atpE protein that could be targeted for vaccine development?

Analysis of the atpE protein reveals several immunogenic epitopes with potential for vaccine development:

• B-cell epitope mapping:

  • Bioinformatic prediction tools (IEDB, ABCpred) identify regions with high surface accessibility, flexibility, and antigenicity

  • The N-terminal region (amino acids 5-20: FISNILANYLGAMSII) shows strong B-cell epitope properties

  • The central region (amino acids 40-55: GLASVGILGTGVGQGL) contains conserved B-cell epitopes

  • These regions show β-turn structures and high hydrophilicity scores

• T-cell epitope analysis:

  • MHC-I and MHC-II binding prediction identifies potential T-cell epitopes

  • The C-terminal region (amino acids 80-95: SAGISESGAIYSLVIA) contains strong T-cell epitope candidates

  • Peptide-binding assays confirm predicted epitopes with IC50 values <500 nM

• Cross-species epitope conservation:

  • Comparison with atpE from M. pneumoniae shows regions of conserved epitopes (amino acids 35-55)

  • Species-specific epitopes exist primarily in the N-terminal region

  • Targeting conserved epitopes could provide broader protection across Mycoplasma species

• Epitope accessibility studies:

  • Membrane topology analysis indicates that certain epitopes (particularly in the loops between transmembrane domains) are surface-exposed

  • These accessible epitopes (amino acids 25-35 and 75-85) represent prime targets for antibody recognition

For effective vaccine design, fusion constructs combining multiple epitopes have shown superior immunogenicity compared to single epitopes in animal models .

What are the optimal storage conditions for maintaining the stability of recombinant atpE protein?

To maintain optimal stability of recombinant atpE protein, the following evidence-based storage protocols are recommended:

• Short-term storage (1-4 weeks):

  • Store at 4°C in buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol

  • Addition of protease inhibitors (PMSF at 1 mM or complete protease inhibitor cocktail)

  • For membrane-associated forms, include 0.03-0.05% DDM detergent

• Long-term storage (months to years):

  • Lyophilized form shows greatest stability at -20°C or -80°C with 12-month shelf life

  • For liquid formulations, store at -80°C in buffer with 50% glycerol

  • Aliquot in small volumes (50-100 μl) to avoid repeated freeze-thaw cycles

  • Typical shelf life is 6 months for liquid form and 12 months for lyophilized form

• Reconstitution protocol:

  • For lyophilized protein, centrifuge vial briefly before opening

  • Reconstitute to 0.1-1.0 mg/mL using deionized sterile water

  • Add glycerol to 5-50% final concentration for storage

  • Avoid repeated freeze-thaw cycles by storing working aliquots at 4°C for up to one week

Stability assessment studies show that recombinant atpE retains >90% activity after 6 months when stored according to these protocols, while improper storage conditions (particularly room temperature storage or repeated freeze-thaw cycles) can reduce activity by 50-70% within weeks .

How can researchers validate the functional activity of recombinant atpE protein?

Validating the functional activity of recombinant atpE requires specific assays that assess its biological properties:

• Proton translocation assay:

  • Reconstitution of purified atpE into liposomes containing pH-sensitive fluorescent dyes (ACMA or pyranine)

  • Application of proton gradient triggers measurable fluorescence changes

  • Active atpE shows characteristic proton conductance patterns

  • Inhibition by DCCD (dicyclohexylcarbodiimide) confirms specificity

• ATP synthase complex reconstitution:

  • Combine recombinant atpE with other ATP synthase subunits from the same or related species

  • Assess ATP hydrolysis/synthesis activity of the reconstituted complex

  • Functional atpE enables measurable ATP synthesis when incorporated into the complex

• Structural integrity assessment:

  • Circular dichroism spectroscopy confirms proper secondary structure (predominantly α-helical)

  • Thermal shift assays determine protein stability (Tm typically 55-65°C for properly folded protein)

  • Size exclusion chromatography verifies oligomeric state

• Binding affinity measurements:

  • Surface plasmon resonance (SPR) to measure interaction with other F0 subunits

  • Isothermal titration calorimetry (ITC) for quantitative binding parameters

  • Active protein shows nanomolar affinity for other ATP synthase components

These approaches collectively provide a comprehensive functional validation, with the reconstitution assays being particularly definitive for confirming biological activity .

How can genetic modification approaches like CRISPR-Cas9 be used to study atpE function in Mycoplasma species?

CRISPR-Cas9 and other genetic modification techniques offer powerful approaches to study atpE function in Mycoplasma species:

• CRISPR-Cas9 genome editing in Mycoplasma:

  • Design sgRNAs targeting atpE gene with minimal off-target effects

  • For Mycoplasma mycoides, optimal PAM sequences (NGG) can be identified within the atpE gene

  • Delivery of Cas9-sgRNA ribonucleoprotein complexes via electroporation (1250 V/25 μF/100 Ω)

  • Use of repair templates with homology arms (500-1000 bp) flanking the target site

  • Screening of edited clones using PCR and sequencing

• Oligonucleotide recombineering approach:

  • Implementation of GP35 (ssDNA recombinase from Bacillus subtilis phage) for efficient recombineering

  • Design of 60-90 nucleotide oligonucleotides matching the lagging strand

  • Introduction of specific mutations in atpE with efficiency rates up to 2.7 × 10^-2

  • Combination with CRISPR-Cas9 counterselection to enrich for edited cells

• Gene knockout/knockdown strategies:

  • Complete knockouts may be lethal due to the essential nature of ATP synthase

  • Conditional expression systems using tetracycline-inducible promoters

  • Partial attenuation using antisense RNA or CRISPRi approaches

  • Phenotypic analysis including growth rate, ATP production, and membrane potential measurements

• Site-directed mutagenesis applications:

  • Introduction of specific mutations in conserved residues (e.g., proton-binding site)

  • Creation of tagged versions for localization studies

  • Engineering of atpE variants with altered inhibitor sensitivity

These genetic approaches have been successfully implemented in Mycoplasma pneumoniae and could be adapted for Mycoplasma mycoides to study atpE function .

What is the role of atpE in Mycoplasma mycoides pathogenicity and how can it be targeted for antimicrobial development?

The role of atpE in Mycoplasma mycoides pathogenicity and its potential as an antimicrobial target involves several dimensions:

• Contribution to pathogenicity:

  • ATP synthase function is essential for energy metabolism and survival during infection

  • Comparative proteomic analysis shows atpE among secreted proteins potentially involved in host-pathogen interactions

  • ATP generation supports virulence factor production, including hydrogen peroxide (H₂O₂) synthesis via the glycerol metabolism pathway

  • Studies suggest ATP synthase components may contribute to membrane integrity and stress responses during infection

• Antimicrobial targeting strategies:

  • Identification of Mycoplasma-specific regions in atpE structure that differ from host ATP synthase

  • Development of specific inhibitors targeting the c-subunit proton channel

  • Screening of small molecule libraries for compounds that disrupt c-ring assembly

  • Peptide-based inhibitors designed to interact with exposed regions of atpE

• Experimental validation approaches:

  • In vitro growth inhibition assays with atpE-targeting compounds

  • Measurement of ATP synthesis in the presence of inhibitors

  • Membrane potential analysis using fluorescent dyes (DiOC₂(3))

  • Assessment of inhibitor specificity against mammalian ATP synthase

• Structure-based drug design:

  • Molecular docking studies using the predicted 3D structure of Mycoplasma mycoides atpE

  • Virtual screening against library of FDA-approved drugs for repurposing

  • Fragment-based drug discovery targeting specific binding pockets

  • Analysis of resistance development through serial passage experiments

Research indicates that targeting atpE could provide a novel strategy against Mycoplasma infections, particularly important given the increasing prevalence of macrolide-resistant strains (MRMP) observed in clinical isolates .

How does atpE interact with other components of the ATP synthase complex in Mycoplasma species?

The interactions between atpE and other ATP synthase components in Mycoplasma species involve complex molecular interactions:

• Structural interactions within the F₀ sector:

  • AtpE subunits form a c-ring oligomer (8-10 subunits) through transmembrane helix interactions

  • Essential interactions occur between the outer helix of atpE and the a-subunit (atpB)

  • These interactions create the critical proton translocation pathway

  • Arginine residues in atpB interact with the proton-carrying glutamate residue in atpE

• Interactions with the F₁ sector:

  • The c-ring interacts with the γ and ε subunits of the F₁ sector

  • These connections allow mechanical coupling between proton movement and ATP synthesis

  • In Mycoplasma species, these interactions appear more direct due to the minimal nature of the ATP synthase complex

• Methodological approaches to study interactions:

  • Crosslinking mass spectrometry (XL-MS) identifies specific interaction sites

  • Co-immunoprecipitation with tagged ATP synthase components

  • Fluorescence resonance energy transfer (FRET) analysis of protein proximity

  • Cryo-EM structural studies of the entire ATP synthase complex

• Species-specific interaction differences:

  • Comparative analysis between M. mycoides and other species (e.g., M. mobile) reveals conserved interaction motifs

  • M. mycoides atpE shows specific sequence adaptations that may influence interactions with other subunits

  • These differences could be exploited for species-specific inhibitor design

Understanding these interactions is crucial for developing targeted antimicrobials and for comprehending the minimal functional requirements of ATP synthases in these metabolically streamlined organisms .

What is the comparative amino acid sequence homology of atpE across different Mycoplasma species?

Table 1: Amino Acid Sequence Identity of atpE Across Selected Mycoplasma Species

What are the key experimental conditions for optimizing expression and purification of recombinant atpE?

Table 2: Optimized Conditions for Expression and Purification of Recombinant Mmm SC atpE

These optimized conditions typically yield 2-5 mg of purified protein per liter of bacterial culture with >90% purity as assessed by SDS-PAGE .

What are the key structural and functional differences between atpE and other ATP synthase subunits in Mycoplasma mycoides?

Table 3: Comparison of ATP Synthase Subunits in Mycoplasma mycoides subsp. mycoides SC