Recombinant Aneura mirabilis ATP synthase subunit C, plastid (atpE)

What is ATP synthase subunit C and what is its role in energy metabolism?

ATP synthase subunit C is an essential component of the F₀ sector of ATP synthase, the enzyme complex responsible for ATP production in cells. This subunit forms part of the membrane-embedded portion (F₀) of the ATP synthase and specifically participates in transmembrane proton conduction. Multiple subunit C proteins assemble into a cylindrical ring structure (C-ring) that rotates during proton translocation through F₀. This rotation is mechanically coupled to the rotation of subunit γ within the α₃β₃ hexamer of the F₁ sector, driving the synthesis of ATP from ADP and inorganic phosphate .

In Aneura mirabilis, a parasitic liverwort, the atpE gene encodes this subunit C in the plastid. This particular protein has gained research interest due to its structural properties and its role in understanding evolutionary adaptations in parasitic plants that have lost photosynthetic capabilities but retained ATP synthase components .

How does the subunit C of ATP synthase differ between Aneura mirabilis and other organisms?

ATP synthase subunit C shows remarkable conservation of function across species despite variations in primary sequence. Comparative analysis of subunit C from different organisms reveals:

The specificity of interactions in the ion-binding site can significantly affect the function and pharmacological targeting of the ATP synthase. For instance, in mycobacteria, a tyrosine specifically conserved at position 64 is part of a cleft between adjacent c subunits that represents a binding site for the drug TMC207, which doesn't affect human ATP synthase . These differences in structure and sequence provide insights into the evolutionary adaptations of ATP synthase in different organisms and can be exploited for specific targeting in research or therapeutic applications.

What expression systems are optimal for producing Recombinant Aneura mirabilis ATP synthase subunit C?

The optimal expression system for producing Recombinant Aneura mirabilis ATP synthase subunit C is E. coli, as evidenced by successful commercial production . When designing an expression system for this membrane protein, researchers should consider:

• Vector selection: pET vectors with T7 promoter systems offer high-level expression for bacterial membrane proteins.

• E. coli strain optimization: BL21(DE3) or its derivatives are preferred due to protease deficiency and T7 RNA polymerase compatibility.

• Expression conditions: Low-temperature induction (16-20°C) often improves proper folding of membrane proteins.

• Solubilization strategy: Given that subunit C is a membrane protein, appropriate detergents for extraction are critical. Research shows that non-ionic detergents are effective for maintaining the structural integrity of subunit C assemblies .

• Fusion tag selection: While commercial preparations use His-tags, researchers might evaluate alternative tags like MBP (maltose-binding protein) for improved solubility or dual tagging strategies for enhanced purification.

Experimental evidence indicates that subunit C can self-assemble into ring structures in non-ionic detergent solutions, even in the absence of other ATP synthase components . This property should be considered when designing expression and purification protocols, as it offers opportunities to study the assembly process in isolation.

What purification methods yield the highest purity and functional integrity of the recombinant protein?

To achieve optimal purification of Recombinant Aneura mirabilis ATP synthase subunit C while maintaining functional integrity, a multi-step purification protocol is recommended:

• Initial extraction: Membrane proteins require careful solubilization. Use of non-ionic detergents such as n-dodecyl β-D-maltoside (DDM) or Triton X-100 at concentrations above their critical micelle concentration is effective for extracting subunit C while preserving its native structure and self-assembly capabilities .

• Affinity chromatography: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA resins provides efficient initial purification. Buffer conditions typically include:

  • Buffer: 50 mM Tris-HCl or sodium phosphate, pH 7.5-8.0

  • Salt: 150-300 mM NaCl to reduce non-specific binding

  • Detergent: 0.05-0.1% DDM or equivalent

  • Imidazole: 10-40 mM for binding, 250-500 mM for elution

• Size exclusion chromatography: This second step separates properly assembled c-rings from monomers and aggregates, providing information about the oligomeric state.

• Quality assessment: Purity evaluation by SDS-PAGE (>90% purity is achievable), followed by functional assessment through reconstitution assays or biophysical characterization .

For storage, the purified protein can be maintained in lyophilized form. When reconstituting, use Tris/PBS-based buffer with 6% trehalose at pH 8.0 to a concentration of 0.1-1.0 mg/mL, with 5-50% glycerol as a cryoprotectant for long-term storage at -20°C/-80°C . Repeated freeze-thaw cycles should be avoided to maintain structural integrity.

How can Recombinant Aneura mirabilis ATP synthase subunit C be used to study self-assembly mechanisms of membrane protein complexes?

Recombinant Aneura mirabilis ATP synthase subunit C provides an excellent model system for investigating fundamental principles of membrane protein self-assembly. Research has demonstrated that subunit c can self-assemble into annular structures in non-ionic detergent solutions without requiring other ATP synthase components . This autonomous assembly property makes it valuable for mechanistic studies.

Methodological approach for self-assembly studies:

• Controlled oligomerization experiments:

  • Purify monomeric subunit C in detergent micelles

  • Manipulate conditions (pH, ionic strength, temperature, lipid composition) to induce oligomerization

  • Monitor assembly kinetics using techniques such as analytical ultracentrifugation, native gel electrophoresis, or light scattering

• Site-directed mutagenesis studies:

  • Introduce mutations at key interface residues to identify critical interaction sites

  • Compare assembly efficiency of wild-type and mutant proteins

  • Correlate findings with structural predictions from homology modeling

• Biophysical characterization of assemblies:

  • Negative-stain electron microscopy or cryo-EM to visualize ring structures

  • Mass spectrometry to determine exact stoichiometry of assemblies

  • Atomic force microscopy to study topography and stability of assembled rings

• Reconstitution into liposomes:

  • Incorporate purified protein into artificial membrane systems

  • Assess functional properties through proton translocation assays

This research direction is particularly valuable because findings from subunit C assembly studies can inform our broader understanding of membrane protein complex formation, potentially aiding the development of novel therapeutic approaches targeting ATP synthase or other membrane protein complexes .

What structural modeling approaches are most effective for studying Aneura mirabilis ATP synthase subunit C interactions?

For effective structural modeling of Aneura mirabilis ATP synthase subunit C interactions, a multi-faceted computational approach informed by experimental data yields the most reliable results:

• Homology modeling pipeline:

  • Template selection: Based on available structures from related organisms like those from I. tartaricus and S. platensis ATP synthase c-rings

  • Sequence alignment optimization: Focus on conserved functional residues such as the ion-binding glutamate

  • Model generation: Using specialized membrane protein modeling tools that account for the lipid bilayer environment

  • Refinement: Molecular dynamics simulations in explicit membrane environments

• Docking simulations for ligand interactions:

  • Receptor preparation: Properly defined protonation states of key residues

  • Identification of binding pockets: Focus on the cleft between adjacent c subunits

  • Evaluation of interactions: Emphasis on ionic, hydrogen, and halogen bonds

• Ring assembly modeling:

  • Oligomer construction: Building the complete c-ring based on monomer models

  • Interface analysis: Evaluation of residue interactions at subunit interfaces

  • Stability assessment: Molecular dynamics simulations of the complete ring

This approach has been successfully applied in studying drug interactions with mycobacterial ATP synthase, where a structure of the C ring constructed by homology modeling was used for docking simulations with the drug TMC207 . The model revealed that a cleft located between two adjacent c subunits, encompassing the proton-binding site (Glu61 in M. tuberculosis), serves as the binding site for the drug, with interactions involving key residues like Glu61, Tyr64, and Asp28 .

For Aneura mirabilis ATP synthase subunit C, similar approaches can identify critical interaction sites for proton binding and subunit-subunit contacts, potentially revealing unique features related to its parasitic lifestyle adaptation.

How does the functional role of ATP synthase in parasitic plant Aneura mirabilis differ from photosynthetic plants?

The functional role of ATP synthase in the parasitic liverwort Aneura mirabilis (also known as Cryptothallus mirabilis) represents a fascinating case of evolutionary adaptation. As a non-photosynthetic parasitic plant, A. mirabilis has undergone significant metabolic remodeling while retaining certain plastid functions:

• Metabolic context differences:

  • Photosynthetic plants: ATP synthase functions primarily in the chloroplast as part of the photosynthetic machinery, utilizing the proton gradient generated by light-driven electron transport

  • A. mirabilis: Has lost photosynthetic capacity but retained ATP synthase, suggesting alternative roles in energy metabolism

• Genome and protein adaptations:

  • Despite its parasitic lifestyle, A. mirabilis has retained the plastid-encoded atpE gene

  • The protein maintains key functional elements like the ion-binding site while potentially having evolved specific adaptations

• Hypothesized alternative functions:

  • Energy acquisition: May function in reverse as an ATPase to generate proton gradients for secondary transport processes

  • Metabolic integration: Could participate in alternative plastid metabolic pathways that remain essential even in parasitic plants

  • Structural role: Might maintain membrane organization in the reduced plastid

A comparative functional analysis examining ATP synthase activity between A. mirabilis and photosynthetic relatives would provide valuable insights into how this complex has been repurposed during the evolution of parasitism. Such research requires careful isolation of plastids from both organism types and comparative biochemical characterization of ATP synthesis/hydrolysis activities under various conditions.

This represents an excellent example of how studying ATP synthase in non-conventional organisms can provide unique evolutionary perspectives on core cellular functions and their adaptability.

What are the common challenges in expressing and purifying functional Recombinant Aneura mirabilis ATP synthase subunit C?

Researchers working with Recombinant Aneura mirabilis ATP synthase subunit C often encounter several technical challenges during expression and purification processes:

• Expression challenges:

  • Toxicity to host cells: Membrane protein overexpression can disrupt host membrane integrity

  • Inclusion body formation: Improper folding leading to protein aggregation

  • Low expression levels: Common with membrane proteins

  Methodological solutions:

  • Implement tightly regulated expression systems with tunable induction

  • Lower induction temperature (16-20°C) and reduce inducer concentration

  • Use specialized E. coli strains like C41(DE3) or C43(DE3) designed for membrane protein expression

  • Co-express with chaperones to improve folding

• Solubilization and purification challenges:

  • Detergent selection: Inappropriate detergents may destabilize the protein structure

  • Maintaining oligomeric state: Preserving native c-ring assembly during purification

  • Detergent-protein-lipid balance: Critical for functional integrity

  Methodological solutions:

  • Screen multiple detergents (DDM, LMNG, Triton X-100) for optimal extraction

  • Include lipids during purification to stabilize protein-protein interactions

  • Apply gentle purification conditions to maintain assembled structures

• Functional validation challenges:

  • Assessing proper folding: Difficult for membrane proteins

  • Evaluation of assembly: Verifying correct c-ring formation

  • Functional reconstitution: Demonstrating proton translocation capability

  Methodological solutions:

  • Circular dichroism to assess secondary structure

  • Negative-stain EM or native PAGE to verify ring assembly

  • Reconstitution into liposomes for functional assays

The evidence from successful purification protocols indicates that non-ionic detergents provide an environment conducive to maintaining subunit C in a state that allows self-assembly into annular structures . Researchers should carefully optimize each step of the process while monitoring protein quality through multiple complementary techniques.

How can researchers effectively study the interaction between Aneura mirabilis ATP synthase subunit C and potential inhibitors?

Studying interactions between Aneura mirabilis ATP synthase subunit C and potential inhibitors requires a systematic approach combining computational, biophysical, and functional methodologies:

• In silico screening and modeling:

  • Develop homology models based on known structures of ATP synthase c-rings

  • Identify potential binding sites through cavity detection algorithms

  • Perform virtual screening of compound libraries against identified binding sites

  • Conduct molecular dynamics simulations to evaluate binding stability

• Biophysical interaction assays:

  • Thermal shift assays: Measure changes in protein thermal stability upon inhibitor binding

  • Surface plasmon resonance (SPR): Determine binding kinetics and affinity constants

  • Isothermal titration calorimetry (ITC): Characterize thermodynamic parameters of binding

  • Microscale thermophoresis: Measure interactions in solution with minimal protein consumption

• Functional validation studies:

  • ATP synthesis assays: Using reconstituted proteoliposomes containing the c-ring

  • Proton translocation measurements: Fluorescence-based assays with pH-sensitive dyes

  • Competitive binding assays: Against known inhibitors or natural substrates

• Structural validation:

  • Hydrogen-deuterium exchange mass spectrometry: Map protein regions involved in binding

  • Cryo-EM analysis: Visualize inhibitor-bound structures at near-atomic resolution

The approach used for studying TMC207 interaction with mycobacterial ATP synthase provides an excellent methodological template . Researchers identified six distinct mutations in subunit c that conferred resistance to TMC207, including changes at positions Asp28, Leu59, Glu61, Ala63, and Ile66 . These findings were integrated with homology modeling and docking simulations to identify a binding cleft between adjacent c subunits .

For Aneura mirabilis ATP synthase subunit C, a similar multidisciplinary approach would enable identification of specific binding sites and potential inhibitors, which could have implications for understanding evolutionary adaptations in ATP synthase and potentially for developing new research tools or therapeutic agents.

What emerging technologies and methodologies hold promise for advancing our understanding of ATP synthase subunit C function?

Several cutting-edge technologies are transforming research on ATP synthase subunit C and hold significant promise for future investigations:

• Advanced structural biology techniques:

  • Single-particle cryo-EM: Allows visualization of membrane proteins in near-native states without crystallization, with recent advances pushing resolution below 2Å

  • Microcrystal electron diffraction (MicroED): Enables structure determination from nanocrystals of membrane proteins

  • Time-resolved crystallography: Captures dynamic states during the catalytic cycle

• Artificial intelligence applications:

  • AI-powered structure prediction: Tools like AlphaFold2 are increasingly accurate for membrane protein prediction

  • Multi-agent AI systems: Google's AI co-scientist approach can analyze complex scientific literature and generate novel hypotheses about ATP synthase function

  • Machine learning-based data integration: Combines structural, functional, and evolutionary data to identify patterns invisible to conventional analysis

• Advanced single-molecule techniques:

  • High-speed atomic force microscopy: Visualizes real-time conformational changes in single ATP synthase molecules

  • Single-molecule FRET: Measures nanoscale distances between labeled components during function

  • Optical tweezers: Quantifies forces involved in c-ring rotation and conformational changes

• Synthetic biology approaches:

  • Minimal ATP synthase systems: Engineered simplified versions to dissect fundamental principles

  • Non-natural amino acid incorporation: Introduces novel chemical properties at specific positions

  • Biomimetic energy systems: Artificial assemblies based on ATP synthase principles

• Multi-omics integration:

  • Combining proteomics, metabolomics, and transcriptomics to understand ATP synthase regulation within cellular networks

  • Systems biology modeling of energy metabolism incorporating ATP synthase function

The integration of AI co-scientist systems with experimental approaches is particularly promising. Such systems can help researchers navigate the extensive scientific literature, identify non-obvious connections between findings in different fields, and generate novel hypotheses about ATP synthase function that might not emerge from conventional research approaches.

How might comparative studies of ATP synthase subunit C across diverse species inform evolutionary understanding and potential biotechnological applications?

Comparative studies of ATP synthase subunit C across diverse species offer profound insights into evolutionary biology and enable novel biotechnological innovations:

• Evolutionary insights from comparative analysis:

  • Adaptation signatures: Comparison between photosynthetic plants and parasitic species like A. mirabilis reveals how ATP synthase adapts during major metabolic transitions

  • Convergent evolution: Identification of independently evolved features for similar functions

  • Co-evolution patterns: Detection of coordinated changes between subunit C and other ATP synthase components

  • Ancestral reconstruction: Inferring the properties of ATP synthase in early life forms

• Methodological approach for comparative studies:

  • Comprehensive phylogenetic sampling across taxonomic diversity

  • Integration of structural, functional, and genomic data

  • Advanced statistical methods to detect selection signatures

  • Experimental validation of evolutionary hypotheses through site-directed mutagenesis

• Biotechnological applications derived from comparative findings:

  • Designer ATP synthases: Engineering custom enzymes with desired properties based on natural diversity

  • Biomimetic energy conversion: Development of synthetic systems inspired by natural variations

  • Targeted antimicrobials: Exploitation of structural differences between bacterial and eukaryotic ATP synthases, similar to the TMC207 example in mycobacteria

  • Biosensors: Utilizing modifications of subunit C for detecting environmental conditions

• Specific research directions:

  • Compare ATP synthase c-rings from extremophiles to understand adaptation to environmental stress

  • Investigate subunit C in organisms with unusual energy metabolism

  • Examine parasitic or symbiotic species that have undergone metabolic streamlining

The discovery that TMC207 specifically targets mycobacterial ATP synthase by binding to a cleft between adjacent c subunits demonstrates how species-specific differences can be exploited . Similar comparative approaches with A. mirabilis and other plant species could reveal unique features with potential biotechnological applications.