Recombinant ATP synthase subunit C, cyanelle (atpE)

What is ATP synthase subunit C from cyanelle and what makes it unique for research?

ATP synthase subunit C from cyanelle (atpE) is a critical component of the F0 sector of ATP synthase in cyanelles, which are peptidoglycan-containing plastids found in glaucocystophyte algae like Cyanophora paradoxa. The protein consists of 81 amino acids and functions as part of the membrane-embedded proton channel that drives ATP synthesis through rotational catalysis.

What makes cyanelle atpE particularly valuable for research is its evolutionary position. Cyanelles represent an intermediate evolutionary stage between cyanobacteria and chloroplasts, offering insights into endosymbiotic processes and the evolution of energy-generating organelles. The protein's structure includes characteristic transmembrane domains that form the c-ring of the F0 complex, making it useful for structural biology and bioenergetic studies .

How should recombinant ATP synthase subunit C, cyanelle (atpE) be stored and reconstituted for optimal activity?

For optimal storage and activity retention, follow these methodological guidelines:

Storage Protocol:

• Store lyophilized protein at -20°C to -80°C upon receipt

• Create working aliquots to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• Long-term storage requires 5-50% glycerol (final concentration) with aliquoting and storage at -20°C/-80°C

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to collect contents at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended as default)

• Aliquot for long-term storage

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

What expression systems are most effective for producing functional recombinant ATP synthase subunit C from cyanelle?

E. coli is the most commonly utilized expression system for recombinant ATP synthase subunit C from cyanelle as demonstrated in commercial preparations . The effectiveness of E. coli stems from several factors:

• The relatively small size of atpE (81 amino acids) makes it amenable to expression in bacterial systems

• His-tagging strategies, particularly N-terminal His tags, maintain protein functionality while facilitating purification

• The hydrophobic nature of this membrane protein requires specialized expression conditions:

  • Reduced induction temperature (16-25°C)

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

  • Specialized media formulations containing glycerol and glucose to regulate expression rates

What techniques are most effective for analyzing the structure and oligomerization state of ATP synthase subunit C from cyanelle?

Recommended Analytical Techniques for Structure and Oligomerization:

For analyzing cyanelle atpE specifically, researchers should note that this protein forms highly stable oligomeric c-rings comprising multiple copies of the subunit C protein. The analysis often requires specialized detergents (DDM, LMNG, or amphipols) to maintain the native oligomeric state during purification and analysis . Cryo-EM has emerged as the preferred method for structural determination of intact ATP synthase complexes due to its ability to resolve membrane proteins in near-native environments.

How does the amino acid sequence of ATP synthase subunit C from cyanelle influence its function and research applications?

The amino acid sequence of ATP synthase subunit C from cyanelle (MDATVSAASVIAAALAVGLAAIGPGIGQGTAAGQAVEGIARQPEVDGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV) contains several functional domains that directly influence its research applications :

• N-terminal region (residues 1-20): Contains targeting information for membrane insertion and assembly

• First transmembrane helix (approximately residues 21-40): Forms part of the c-ring structure

• Loop region (approximately residues 41-50): Contains conserved charged residues important for interaction with other ATP synthase subunits

• Second transmembrane helix (approximately residues 51-70): Houses the critical proton-binding glutamate residue

• C-terminal region (residues 71-81): Important for stability and assembly

These sequence features enable several specialized research applications:

• Proton binding and energy transduction studies: The conserved glutamate residue in the second transmembrane helix is the site of protonation/deprotonation driving the rotary mechanism

• Evolutionary studies: Comparative analysis with bacterial, chloroplast, and mitochondrial ATP synthase c subunits provides insights into organelle evolution

• Structural biology: The small size and defined secondary structure make it useful for membrane protein structure studies

• Drug development: The c-subunit is a target for certain antibiotics and inhibitors, making it valuable for structure-based drug design

Mutations in key residues have been used to study the mechanism of proton translocation and coupling to ATP synthesis, providing fundamental insights into bioenergetic principles.

How can ATP synthase subunit C from cyanelle be utilized in comparative genomic and evolutionary studies?

ATP synthase subunit C from cyanelle serves as an excellent molecular marker for evolutionary biology research due to its position at the interface of prokaryotic and eukaryotic systems. To effectively utilize this protein in comparative genomics:

• Phylogenetic Analysis Protocol:

  • Extract atpE sequences from cyanelles, cyanobacteria, chloroplasts, and mitochondria

  • Perform multiple sequence alignment using MUSCLE or MAFFT algorithms

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Analyze conserved motifs and evolutionary rates to trace endosymbiotic events

• Synteny Analysis:

  • Compare genomic context of atpE across organisms with complete plastid genomes

  • Map gene order and arrangement as shown in chloroplast genomes like Chlamydomonas

  • Identify genomic rearrangements indicating evolutionary transitions

• Selective Pressure Analysis:

  • Calculate dN/dS ratios to identify sites under positive or purifying selection

  • Compare substitution rates between cyanelles and other plastid types

  • Correlate conservation patterns with structural features

This approach has revealed that cyanelle atpE retains characteristics of both cyanobacterial ancestors and more derived chloroplasts, with particular conservation patterns in the proton-binding regions. The evolutionary position of cyanelles as "living fossils" of early endosymbiotic events makes their ATP synthase components particularly valuable for tracing the evolution of organellar energy-generating systems .

What are the methodological approaches for studying protein-protein interactions between ATP synthase subunit C and other components of the ATP synthase complex?

Studying the protein-protein interactions of ATP synthase subunit C requires specialized approaches due to its hydrophobic nature and integration within a multi-subunit membrane complex. The following methodological approaches are recommended:

In vitro Interaction Studies:

• Co-immunoprecipitation with Membrane Protein Adaptations:

  • Solubilize membranes with mild detergents (DDM, digitonin)

  • Use crosslinking with DSP or formaldehyde prior to solubilization

  • Perform pull-down with antibodies against atpE or tagged versions

  • Validate interactions with western blotting and mass spectrometry

• Microscale Thermophoresis (MST):

  • Label purified atpE with fluorescent dyes

  • Measure thermophoretic movement in response to binding partners

  • Determine binding affinities and kinetics in near-native conditions

• Surface Plasmon Resonance (SPR):

  • Immobilize reconstituted atpE in nanodiscs or supported lipid bilayers

  • Measure real-time binding of other ATP synthase components

  • Quantify association/dissociation constants

In vivo Interaction Studies:

• Split-GFP Complementation:

  • Fuse fragments of GFP to atpE and potential interacting partners

  • Express in appropriate model systems

  • Visualize interaction through reconstituted GFP fluorescence

• FRET-based Assays:

  • Tag atpE and interacting partners with appropriate FRET pairs

  • Measure energy transfer efficiency to confirm proximity

  • Use time-resolved FRET to study dynamic interactions

These methodologies have revealed that cyanelle atpE interacts primarily with other c-subunits to form the c-ring, as well as with the a-subunit that forms the stationary part of the proton channel, and peripherally with subunits b and δ of the ATP synthase stator.

How do structural variations in ATP synthase subunit C across different plastid types affect enzymatic function and research applications?

Structural variations in ATP synthase subunit C across different plastid types have profound implications for both enzymatic function and research applications. Comparative analysis reveals:

Structural Variations and Functional Implications:

The structure of cyanelle atpE represents an evolutionary intermediate with unique properties:

• The protein retains features of both cyanobacterial ancestors and derived chloroplasts

• The c-ring typically contains 14 subunits, compared to 8-15 in other systems

• These structural differences affect the H⁺/ATP ratio and thermodynamic efficiency

Researchers can exploit these differences to study how structural variations impact:

• The bioenergetic cost of ATP synthesis

• Adaptation to different environmental conditions

• Evolution of organellar energy transduction mechanisms

• Development of synthetic biology applications with customized energy parameters

The variation in c-ring stoichiometry across species directly influences the thermodynamic H⁺/ATP ratio, making comparative studies of atpE structure particularly valuable for understanding bioenergetic adaptation.

What are the common challenges in expressing and purifying functional ATP synthase subunit C from cyanelle, and how can researchers overcome them?

Researchers face several recurring challenges when working with ATP synthase subunit C from cyanelle due to its hydrophobic nature and tendency to form oligomeric structures. The following table outlines these challenges and provides methodological solutions:

Advanced researchers should note that atpE from cyanelle requires specialized approaches compared to other ATP synthase components:

• The protein has high hydrophobicity requiring detergent concentrations 2-3× higher than typical membrane proteins

• Functional studies often require reconstitution of the entire c-ring, not just monomeric subunits

• The reconstitution buffer should match cyanelle physiological conditions (pH 7.0-7.5) rather than standard chloroplast conditions

These optimized protocols significantly improve both yield and functionality of the recombinant protein for downstream structural and functional studies.

How can researchers effectively design experiments to compare the functional properties of ATP synthase subunit C from different evolutionary sources?

Designing rigorous comparative studies of ATP synthase subunit C requires careful experimental planning to account for evolutionary differences while maintaining methodological consistency. The following framework provides a comprehensive approach:

Experimental Design Protocol for Evolutionary Comparison:

• Sample Selection and Preparation:

  • Select representative species spanning evolutionary lineages (cyanobacteria, cyanelles, chloroplasts, mitochondria)

  • Express proteins using identical tags and expression systems where possible

  • Purify using standardized protocols with appropriate modifications for specific proteins

  • Reconstitute in identical lipid compositions (e.g., 70:30 POPE:POPG)

• Functional Assays with Standardized Parameters:

  • ATP synthesis rate measurement:

    • Use identical pH gradients (typically ΔpH of 3.0)

    • Maintain consistent temperature (25°C)

    • Measure ATP production using luciferase-based assays

    • Calculate and compare ATP synthesis rates

  • Proton conductance measurement:

    • Reconstitute proteins in liposomes loaded with pH-sensitive dyes

    • Apply identical membrane potentials

    • Record proton flux rates under controlled conditions

    • Compare coupling ratios (H⁺/ATP)

• Structural Comparison:

  • Determine c-ring stoichiometry using AFM or electron microscopy

  • Compare 3D structures at equivalent resolutions

  • Analyze conserved and variable regions using computational methods

• Data Normalization and Statistical Analysis:

  • Normalize all functional data to protein concentration

  • Apply appropriate statistical tests for multiple comparisons

  • Use ANOVA with post-hoc tests to identify significant differences

  • Calculate effect sizes to quantify evolutionary divergence

• Controls for Experimental Validation:

  • Include hybrid constructs with domain swapping between species

  • Test under varying physiological conditions relevant to each organism

  • Validate with in vivo complementation assays where possible

This approach has revealed that cyanelle ATP synthase subunit C exhibits functional properties intermediate between those of cyanobacteria and chloroplasts, particularly in proton affinity and rotational coupling, providing important insights into the evolutionary adaptation of bioenergetic systems.

What are the most effective methods for analyzing the role of ATP synthase subunit C in the context of complete chloroplast genome studies?

Analyzing ATP synthase subunit C within the broader context of chloroplast genomics requires integrated approaches that connect gene-level analyses with genome-wide perspectives. Based on methodologies used in comprehensive studies like those of Chlamydomonas reinhardtii , researchers should implement the following strategies:

These approaches have revealed that atpE is located within conserved gene clusters in many plastid genomes, often co-transcribed with other ATP synthase components, though specific arrangements vary across evolutionary lineages. In Chlamydomonas, the circular and linear forms of the chloroplast genome add complexity to gene expression and inheritance patterns , affecting the regulation of energy-generating components like ATP synthase.

Short dispersed repeats (SDRs) found in many chloroplast genomes may influence atpE expression and evolution, as these repetitive elements can facilitate recombination events and genome rearrangements. The application of advanced cytogenomic approaches, including fiber-FISH techniques as demonstrated in Chlamydomonas studies , provides powerful tools for visualizing and analyzing the genomic context of key genes like atpE in various plastid types.

What emerging technologies and methodologies show promise for advancing our understanding of ATP synthase subunit C from cyanelle?

Several cutting-edge technologies are poised to revolutionize research on ATP synthase subunit C from cyanelle, opening new avenues for structural, functional, and evolutionary investigations:

• Cryo-Electron Tomography:

  • Enables visualization of ATP synthase in its native membrane environment

  • Reveals native stoichiometry and supramolecular organization

  • Potential to capture different conformational states during rotation

• Time-Resolved Serial Femtosecond Crystallography:

  • Captures transient structural states during the catalytic cycle

  • Provides dynamic information about proton binding and conformational changes

  • Requires specialized expression and crystallization of membrane proteins

• Single-Molecule FRET and Force Spectroscopy:

  • Directly measures rotational dynamics of individual ATP synthase molecules

  • Quantifies force generation and energy transduction at the molecular level

  • Requires sophisticated fluorescent labeling strategies for membrane proteins

• Artificial Intelligence and Deep Learning Applications:

  • Improves protein structure prediction specific to membrane proteins like atpE

  • Enables in silico modeling of protein dynamics and interactions

  • Facilitates design of optimized atpE variants with enhanced properties

• CRISPR-Based Chloroplast Genome Editing:

  • Allows precise manipulation of atpE in its native genomic context

  • Enables creation of custom mutations to probe structure-function relationships

  • Provides tools for synthetic biology applications in chloroplasts

These emerging technologies will help address fundamental questions about cyanelle ATP synthase subunit C that remain unanswered, particularly regarding the molecular details of proton translocation, the evolutionary advantages of different c-ring stoichiometries, and the potential for engineering optimized ATP synthase variants for biotechnological applications.

What are the key considerations for researchers designing comprehensive studies of ATP synthase subunit C from cyanelle?

Researchers embarking on studies of ATP synthase subunit C from cyanelle should consider several critical factors to ensure robust and meaningful outcomes:

• Evolutionary Context:

  • Position cyanelle studies within the broader evolutionary landscape of plastid development

  • Compare with both ancestral (cyanobacterial) and derived (chloroplast) systems

  • Consider the unique evolutionary position of Cyanophora paradoxa as a model for early endosymbiosis

• Methodological Integration:

  • Combine structural studies (cryo-EM, X-ray crystallography) with functional assays

  • Incorporate genomic context from complete plastid genome analyses

  • Link molecular-level findings to organelle and organism-level physiology

• Technical Challenges:

  • Address the inherent difficulties of membrane protein biochemistry

  • Develop specialized protocols for expression, purification, and reconstitution

  • Consider the impact of experimental conditions on native structure and function

• Future Applications:

  • Explore potential biotechnological applications in synthetic biology and bioengineering

  • Investigate the role of ATP synthase in environmental adaptation

  • Develop cyanelle-based systems for bioenergetic research