Recombinant Calycanthus floridus var. glaucus ATP synthase subunit c, chloroplastic (atpH)

What is the biological role of ATP synthase subunit c, chloroplastic (atpH) in Calycanthus floridus var. glaucus?

ATP synthase subunit c is a critical component of the F0 sector of ATP synthase in chloroplasts. It functions as part of the c-ring within the F0 complex, which serves as the main constituent of the rotor in the ATP synthesis machinery. This transmembrane protein forms oligomeric rings that facilitate proton translocation across the membrane, driving the conformational changes necessary for ATP synthesis. In Calycanthus floridus var. glaucus (Eastern sweetshrub), as in other plants, this protein plays an essential role in energy conversion within chloroplasts by harnessing the proton gradient established during photosynthesis .

What are the optimal storage conditions for working with this recombinant protein?

For optimal stability and activity retention, the recombinant ATP synthase subunit c should be stored according to these guidelines:

• Long-term storage: -20°C to -80°C

• Working aliquots: 4°C for up to one week

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

• For liquid formulations, shelf life is approximately 6 months at -20°C/-80°C

• For lyophilized formulations, shelf life extends to 12 months at -20°C/-80°C

• Storage buffer typically consists of Tris-based buffer with 50% glycerol optimized for protein stability

To maximize shelf life, it is recommended to prepare small working aliquots to minimize exposure to freeze-thaw cycles, which can lead to protein degradation and loss of structural integrity.

What reconstitution methods are recommended for experimental applications?

The following methodological approach is recommended for reconstitution:

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

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

• Add glycerol to a final concentration of 5-50% (typically 50% is used as standard)

• Prepare small aliquots for long-term storage at -20°C/-80°C

• For membrane protein functional studies, consider incorporating the protein into liposomes or nanodiscs to maintain native conformation

This methodological approach maintains protein stability while allowing flexibility for different experimental applications, from structural studies to functional assays.

How is protein purity assessed for research applications?

For research applications requiring high-quality protein preparations, purity assessment is typically performed using:

• SDS-PAGE analysis, with quality preparations showing >85-90% purity

• Size exclusion chromatography to assess oligomeric state and homogeneity

• Mass spectrometry to confirm protein identity and detect potential modifications

• Circular dichroism to verify proper secondary structure formation

• Functional assays to confirm biological activity

These analytical methods ensure that the recombinant protein meets the rigorous standards required for downstream research applications, particularly for structural studies or enzyme kinetics experiments.

What experimental approaches are optimal for studying the oligomerization properties of ATP synthase subunit c?

Investigating the oligomerization of ATP synthase subunit c requires multiple complementary approaches:

• Atomic Force Microscopy (AFM): Enables visualization of c subunit ultrastructure and assembly into oligomeric complexes under different conditions. Samples should be pre-incubated at 37°C and deposited on mica for imaging .

• ThT Fluorescence Assay: Measures the kinetics of protein aggregation and fibril formation. Protein samples (typically 5-10 μM) are incubated with Thioflavin T (10-20 μM) at 37°C, with fluorescence measurements taken at regular intervals (exc. 440 nm, em. 480 nm). This assay can reveal the effects of factors like calcium on aggregation kinetics .

• Gel Electrophoresis: Native PAGE and BN-PAGE can identify oligomeric species ranging from 15 to 250 kDa. SDS-resistant oligomers can be detected using SDS-PAGE without boiling samples .

• Crosslinking Studies: Chemical crosslinkers (e.g., glutaraldehyde, DSS) can capture transient oligomeric states for subsequent analysis by electrophoresis and mass spectrometry.

• Analytical Ultracentrifugation: Determines the distribution of oligomeric species in solution under native conditions.

The integration of these methodologies provides comprehensive insights into the complex oligomerization behavior of the c subunit under physiologically relevant conditions.

How can researchers investigate the calcium-dependent properties of ATP synthase subunit c?

The calcium-dependent properties of ATP synthase subunit c can be systematically investigated using the following methodological framework:

• ThT Fluorescence Assays: Compare aggregation kinetics in calcium-free versus calcium-containing buffers (typically 1 mM Ca²⁺). Research indicates that calcium strongly suppresses fibril formation, suggesting complex regulatory roles .

• Spectroscopic Methods: Circular dichroism spectroscopy can detect calcium-induced conformational changes, particularly transitions between α-helical and β-sheet structures.

• Calcium Binding Assays: Isothermal titration calorimetry (ITC) and microscale thermophoresis can determine calcium binding parameters, including affinity constants and binding stoichiometry.

• Membrane Permeabilization Assays: Using liposomes loaded with calcium-sensitive fluorophores to investigate calcium-induced permeability transition mediated by the c subunit.

• Structural Analysis: X-ray crystallography or cryo-EM studies in the presence and absence of calcium can reveal the molecular basis of calcium regulation.

This calcium-dependent behavior may have significant implications for understanding both physiological functions and pathological roles of the c subunit in membrane permeabilization and permeability transition .

What techniques are most effective for studying the membrane integration of this transmembrane protein?

To effectively study the membrane integration of ATP synthase subunit c, researchers should consider these methodological approaches:

• Reconstitution into Model Membranes:

  • Liposomes of defined lipid composition (typically phosphatidylcholine/phosphatidylethanolamine mixtures)

  • Nanodiscs with MSP1D1 scaffold proteins

  • Bicelles for NMR applications

• Biophysical Characterization:

  • Fluorescence spectroscopy with site-specific labels to track membrane insertion

  • Tryptophan fluorescence to monitor hydrophobic environment changes

  • FRET assays to measure protein-lipid interactions

  • Infrared spectroscopy to determine secondary structure in membrane environments

• Functional Assays:

  • Black lipid membrane (BLM) conductance measurements

  • Patch-clamp studies of reconstituted proteoliposomes

  • Proton translocation assays using pH-sensitive dyes

• Computational Approaches:

  • Molecular dynamics simulations of membrane insertion and oligomerization

  • Hydrophobicity analysis to predict membrane-spanning regions

• Structural Methods:

  • Solid-state NMR for high-resolution structural information in the membrane

  • Cryo-electron microscopy of membrane-reconstituted complexes

These combined approaches provide a comprehensive understanding of how the highly hydrophobic α-helical hairpin structure of ATP synthase subunit c integrates into and functions within the lipid bilayer environment .

How can researchers study the amyloidogenic properties of ATP synthase subunit c?

The study of amyloidogenic properties of ATP synthase subunit c requires specialized techniques and experimental designs:

• Amyloid Detection Assays:

  • Thioflavin T fluorescence kinetics (monitoring at 480 nm after excitation at 440 nm)

  • Congo Red binding with characteristic green birefringence under polarized light

  • 8-anilino-1-naphthalenesulfonic acid (ANS) binding to detect hydrophobic surfaces

• Structural Characterization of Fibrils:

  • Atomic Force Microscopy (AFM) for visualization of fibril morphology

  • Transmission Electron Microscopy with negative staining

  • X-ray fiber diffraction to confirm cross-β sheet structure

• Monitoring β-sheet Formation:

  • Circular dichroism spectroscopy to track α-helix to β-sheet transitions

  • FTIR spectroscopy focusing on the amide I band (1600-1700 cm⁻¹)

  • Solid-state NMR to determine molecular structure of fibrils

• Seeding Experiments:

  • Using preformed fibrils to accelerate aggregation

  • Cross-seeding with other amyloidogenic proteins to assess specificity

• Environmental Factors Assessment:

  • pH dependence of fibril formation

  • Effect of calcium and other ions on aggregation kinetics

  • Temperature influence on aggregation pathways

Research has shown that ATP synthase subunit c can spontaneously fold into β-sheets and self-assemble into fibrils in a calcium-dependent manner, with calcium ions (1mM) strongly suppressing fibril formation. This suggests complex regulatory mechanisms that may be relevant to both physiological functions and pathological conditions .

What methodological considerations are important when investigating the role of ATP synthase subunit c in permeability transition?

Investigating the role of ATP synthase subunit c in permeability transition requires careful methodological considerations:

• Isolation and Preparation of Mitochondria:

  • Gentle isolation procedures to maintain mitochondrial integrity

  • Quality control assessing respiratory control ratios

  • Preparation of sub-mitochondrial particles for specific assays

• Permeability Transition Assays:

  • Calcium retention capacity measurements with calcium-sensitive dyes

  • Mitochondrial swelling assays monitoring absorbance at 540 nm

  • Membrane potential measurements using potentiometric dyes (TMRM, JC-1)

  • Cytochrome c release detection by Western blotting or ELISA

• Manipulation of c Subunit Expression/Function:

  • RNA interference or CRISPR/Cas9 for genetic knockdown/knockout

  • Site-directed mutagenesis of key residues

  • Chemical inhibitors specific to the c subunit

  • Antibodies that target specific epitopes on the c subunit

• Reconstitution Experiments:

  • Black lipid membrane conductance with purified c subunit

  • Proteoliposomes with reconstituted c subunit to assess ion permeability

  • Patch-clamp studies of reconstituted channels

• Calcium Dependence Assessment:

  • Titration of calcium concentrations (typically 0-1mM range)

  • Use of calcium chelators (EGTA, BAPTA) as controls

  • Co-factors that may modulate calcium sensitivity

These approaches help distinguish between the physiological role of the c subunit in ATP synthesis and its potential pathological role in calcium-induced permeability transition, which has been implicated in mitochondrial dysfunction in various disease states .

What are the best expression systems and purification strategies for obtaining high-quality recombinant ATP synthase subunit c?

Obtaining high-quality recombinant ATP synthase subunit c for structural and functional studies requires optimized expression and purification strategies:

• Expression Systems:

  • E. coli BL21(DE3) with C41/C43 derivatives optimized for membrane proteins

  • Baculovirus-insect cell systems for eukaryotic post-translational modifications

  • Cell-free expression systems for difficult-to-express membrane proteins

• Expression Optimization:

  • Codon optimization for the expression host

  • Fusion partners (His-tag, MBP, SUMO) to enhance solubility

  • Reduced temperature expression (16-20°C) to improve folding

  • Inducer concentration titration (IPTG typically 0.1-0.5 mM)

• Purification Strategy:

  • Membrane isolation by differential centrifugation

  • Detergent solubilization screening (DDM, LDAO, Fos-choline)

  • IMAC purification using His-tag (Ni-NTA or TALON resins)

  • Size exclusion chromatography for oligomeric state separation

  • Ion exchange chromatography for final polishing

• Quality Assessment:

  • SDS-PAGE with Coomassie staining (>90% purity)

  • Mass spectrometry for identity confirmation

  • Circular dichroism for secondary structure verification

  • Functional reconstitution assays

Based on the search results, researchers have successfully expressed the full-length Calycanthus floridus var. glaucus ATP synthase subunit c in E. coli with N-terminal His-tag, achieving purities greater than 90% as determined by SDS-PAGE .

How does the structure and function of ATP synthase subunit c from Calycanthus floridus compare to other plant species?

The ATP synthase subunit c from Calycanthus floridus var. glaucus can be analyzed in an evolutionary context through comparative studies:

• Sequence Alignment Analysis:

  • Multiple sequence alignment of c subunits from diverse plant species

  • Identification of conserved residues essential for function

  • Mapping of species-specific variations that may relate to environmental adaptations

• Structural Comparison Methodologies:

  • Homology modeling based on available high-resolution structures

  • Molecular dynamics simulations to compare dynamic properties

  • Conservation mapping onto structural models to identify functional regions

• Functional Comparative Assays:

  • Reconstitution of c subunits from different species into liposomes

  • Comparative proton translocation efficiency measurements

  • Oligomerization pattern analysis across species

• Evolutionary Rate Analysis:

  • Calculation of dN/dS ratios to identify selection pressures

  • Reconstruction of ancestral sequences to trace evolutionary trajectories

  • Correlation of sequence changes with environmental adaptations

The 81-amino acid sequence of Calycanthus floridus var. glaucus ATP synthase subunit c represents a highly conserved protein, with the transmembrane α-helical hairpin structure being particularly well preserved across species due to its fundamental role in energy conversion .

What experimental approaches can detect post-translational modifications of ATP synthase subunit c?

Detecting and characterizing post-translational modifications (PTMs) of ATP synthase subunit c requires sophisticated analytical approaches:

• Mass Spectrometry-Based Methods:

  • High-resolution LC-MS/MS with CID, HCD, or ETD fragmentation

  • MALDI-TOF MS for intact mass determination

  • Top-down proteomics for comprehensive PTM mapping

  • Targeted MRM assays for specific modifications

• Enrichment Strategies:

  • Phosphopeptide enrichment using TiO₂ or IMAC

  • Immunoprecipitation with modification-specific antibodies

  • Chemical labeling strategies for specific PTMs

• Site-Specific Mutational Analysis:

  • Alanine scanning of potential modification sites

  • Phosphomimetic mutations (S/T to D/E)

  • Non-modifiable mutations (K to R for ubiquitination sites)

• Functional Impact Assessment:

  • Activity assays comparing native and demodified protein

  • Structural analysis of modification effects on conformation

  • Oligomerization assessment with and without modifications

• Cellular Localization Studies:

  • Immunofluorescence with modification-specific antibodies

  • Subcellular fractionation and Western blotting

  • Pulse-chase experiments to track modification dynamics

These methodologies allow researchers to determine how PTMs regulate ATP synthase subunit c function, potentially influencing its dual roles in ATP synthesis and membrane permeabilization .

What are the most effective methods for studying protein-protein interactions involving ATP synthase subunit c?

Investigating protein-protein interactions of ATP synthase subunit c requires specialized approaches suitable for membrane proteins:

• Crosslinking Technologies:

  • Chemical crosslinking with BS³, DSS, or formaldehyde

  • Photo-reactive crosslinkers for temporal control

  • Site-specific incorporation of photo-crosslinkable amino acids

  • Crosslink identification by mass spectrometry (XL-MS)

• Co-Immunoprecipitation Adaptations:

  • Detergent screening for optimal solubilization while preserving interactions

  • Mild solubilization using digitonin or amphipols

  • Pull-down assays with tagged recombinant proteins

  • Reverse co-IP to confirm interactions

• Biophysical Interaction Measurements:

  • Surface plasmon resonance (SPR) with detergent-solubilized or nanodisc-reconstituted proteins

  • Microscale thermophoresis for detecting interactions in complex solutions

  • Fluorescence-based assays (FRET, FCCS) for detecting complex formation

• Advanced Microscopy:

  • FRET/FLIM to visualize interactions in membranes

  • Proximity ligation assay for detecting interactions in situ

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization

• Functional Reconstitution:

  • Co-reconstitution of interacting partners in liposomes

  • Activity assays in the presence or absence of binding partners

  • Competition assays to identify interaction sites

These methods help identify the extensive interaction network of ATP synthase subunit c, including interactions within the ATP synthase complex and potentially with other cellular components, particularly in pathological conditions .

How can researchers effectively reconstruct the ATP synthase complex for structural studies?

Reconstructing the ATP synthase complex for structural studies requires a systematic approach:

• Component Expression and Purification:

  • Individual subunit expression with compatible tags

  • Co-expression strategies for stable subcomplexes

  • Sequential purification of components with orthogonal tags

  • Quality control of each component before assembly

• Complex Assembly Methodologies:

  • In vitro reconstitution from purified components

  • Co-expression of multiple subunits in appropriate stoichiometry

  • Cell-free expression systems for direct complex assembly

  • Gradual detergent removal for membrane protein complexes

• Structural Stabilization:

  • Amphipol or nanodisc incorporation for membrane components

  • Chemical crosslinking to maintain complex integrity

  • Antibody fragment (Fab) binding to stabilize flexible regions

  • Engineered disulfide bonds to lock conformational states

• Structural Analysis Techniques:

  • Cryo-electron microscopy for high-resolution structures

  • X-ray crystallography of stabilized complexes

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

  • Integrative modeling combining multiple data sources

• Functional Validation:

  • ATP synthesis/hydrolysis assays of reconstituted complex

  • Proton pumping measurements in proteoliposomes

  • Rotational analysis using single-molecule techniques

These methodological approaches facilitate the structural characterization of the ATP synthase complex, providing insights into how the c subunit from Calycanthus floridus var. glaucus integrates into the larger ATP synthase machinery and contributes to its function .

What are common challenges in working with recombinant ATP synthase subunit c and how can they be addressed?

Working with ATP synthase subunit c presents several technical challenges that can be addressed through specific methodological approaches:

• Protein Aggregation Issues:

  • Challenge: Tendency to form aggregates during purification

  • Solutions:

    • Addition of mild detergents (0.03-0.1% DDM)

    • Inclusion of glycerol (5-50%) in all buffers

    • Working at lower protein concentrations (<1 mg/mL)

    • Maintaining low temperature during purification (4°C)

• Solubility and Refolding Problems:

  • Challenge: Maintaining proper folding of this hydrophobic protein

  • Solutions:

    • Screening multiple detergents (DDM, LDAO, Fos-choline-12)

    • Testing different reconstitution methods (direct dilution, dialysis)

    • Inclusion of lipids during refolding

    • Stepwise reduction of denaturant concentration

• Protein Quantification Difficulties:

  • Challenge: Accurate concentration determination

  • Solutions:

    • Amino acid analysis as gold standard

    • BCA assay with appropriate detergent controls

    • UV absorption with calculated extinction coefficient

    • SDS-PAGE with standard curve comparison

• Storage Stability Issues:

  • Challenge: Activity loss during storage

  • Solutions:

    • Store at -80°C in small aliquots

    • Include glycerol (50%) in storage buffer

    • Avoid repeated freeze-thaw cycles

    • Consider lyophilization for long-term storage

• Functional Assay Development:

  • Challenge: Measuring specific activity

  • Solutions:

    • Reconstitution into proteoliposomes for function

    • Developing specific antibodies for detection

    • Fluorescent labeling at non-critical residues

    • Complementation assays in knockout systems

Following these technical recommendations enables researchers to overcome the inherent challenges of working with this highly hydrophobic membrane protein and obtain reliable experimental results.

How can researchers validate the functional integrity of recombinant ATP synthase subunit c?

Validating the functional integrity of recombinant ATP synthase subunit c requires multiple complementary approaches:

• Structural Integrity Assessment:

  • Circular dichroism to confirm α-helical content

  • Intrinsic tryptophan fluorescence to verify proper folding

  • Limited proteolysis to assess compact structure

  • Dynamic light scattering to evaluate homogeneity

• Membrane Integration Tests:

  • Flotation assays with liposomes

  • Protease protection assays in membrane environments

  • Fluorescence quenching with membrane-impermeable quenchers

  • FRET-based assays for insertion into lipid bilayers

• Oligomerization Analysis:

  • Native gel electrophoresis for oligomeric state

  • Size exclusion chromatography with multi-angle light scattering

  • Analytical ultracentrifugation to determine assembly state

  • Electron microscopy to visualize c-ring formation

• Functional Reconstitution:

  • Proton translocation assays using pH-sensitive dyes

  • Incorporation into ATP synthase depleted of c subunits

  • Measurement of ATP synthesis in reconstituted systems

  • Patch-clamp analysis of ion channel activity

• Binding Partner Interactions:

  • Pull-down assays with other ATP synthase subunits

  • Surface plasmon resonance to measure binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

  • Co-reconstitution with partner proteins

These validation methods ensure that the recombinant protein maintains native-like properties essential for meaningful experimental outcomes in structural and functional studies .