Recombinant Guillardia theta ATP synthase subunit c, chloroplastic (atpH)
Description
Recombinant Production and Purification
Recombinant atpH is synthesized in Escherichia coli using optimized codon sequences to enhance expression. Key steps include:
-
Cloning: The atpH gene is inserted into expression vectors with flanking restriction sites.
-
Expression: The protein is often produced as a maltose-binding protein (MBP) fusion to improve solubility .
-
Purification:
Mechanistic Studies
Recombinant atpH enables structural and functional analysis of the c-ring:
-
C-Ring Stoichiometry: Experiments with recombinant c-subunits aim to resolve how subunit number (n) influences proton-to-ATP ratios .
-
Membrane Protein Folding: Studies investigate how hydrophobic segments fold into α-helices during insertion into lipid bilayers .
Evolutionary Context
Phylogenetic studies of chloroplast genomes (e.g., Guillardia theta) reveal conserved gene clusters, including atpH, indicating ancestral origins in cyanobacteria .
Challenges and Future Directions
-
Structural Reconstitution: Recombinant c-subunits are used to reassemble c-rings in vitro, but challenges remain in mimicking native lipid environments .
-
Functional Variability: Differences in c-subunit number (n) across species (e.g., 14 in spinach vs. other organisms) require further investigation using recombinant models .
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific requirements for the format, please indicate them in your order remarks. We will prepare the product accordingly.
Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please inform us in advance as additional charges will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
Q&A
What is Guillardia theta ATP synthase subunit c and what is its role in chloroplasts?
Guillardia theta ATP synthase subunit c (atpH) is a key component of the F(0) channel in the F-type ATP synthase complex located in chloroplasts. This protein plays a direct and essential role in proton translocation across the membrane. The F(1)F(0) ATP synthase produces ATP from ADP in the presence of a proton gradient, with the F(1) domain containing the extramembraneous catalytic core and F(0) containing the membrane proton channel. These domains are linked by central and peripheral stalks. During catalysis, ATP synthesis in the F(1) domain is coupled to proton translocation via a rotary mechanism involving the central stalk subunits .
The subunit c forms a homomeric c-ring structure comprising between 10-14 subunits, which constitutes the central stalk rotor element together with the F(1) delta and epsilon subunits. This ring structure is critical for the rotational catalysis mechanism that drives ATP synthesis .
How can recombinant Guillardia theta ATP synthase subunit c be expressed and purified for research purposes?
Expression and purification of recombinant Guillardia theta ATP synthase subunit c presents several challenges due to its highly hydrophobic nature and membrane-spanning domains. Based on methodologies applied to similar proteins, the following protocol can be employed:
-
Expression system selection: E. coli BL21(DE3) strain has been successfully used for expressing similar proteins from Guillardia theta. Culture conditions typically involve growth at 37°C in LB medium supplemented with 100 mM sorbitol and 2.5 mM betaine to an OD578 of 0.6-0.8 .
-
Vector and induction considerations: pGEX-6P1 or pASK-IBA7+ expression vectors can be utilized, with induction using either isopropyl β-D-thiogalactopyranoside (0.5 mM) or anhydrotetracycline (200 ng/ml), respectively .
-
Co-expression with chaperones: To enhance proper folding, co-expression with chaperones such as GroEL and GroES (using pGro7 from TaKaRa) can be induced with 0.5 mg/ml L-arabinose .
-
Incubation conditions: After induction, optimal protein expression is typically achieved by overnight incubation at 17°C to minimize inclusion body formation and maximize functional protein yield .
-
Purification strategy: Due to its hydrophobic nature, detergent-based extraction followed by affinity chromatography is recommended. For GST-tagged constructs, glutathione affinity purification can be employed, followed by tag removal if necessary.
This expression and purification protocol provides a starting point that may require optimization depending on specific experimental requirements and downstream applications.
What methods are used to verify the correct folding and function of recombinant Guillardia theta ATP synthase subunit c?
Verification of correct folding and function of recombinant Guillardia theta ATP synthase subunit c involves multiple complementary approaches:
-
Circular Dichroism (CD) Spectroscopy: This technique can confirm the predominant α-helical secondary structure expected for the correctly folded protein. The α-helical hairpin conformation of ATP synthase subunit c produces characteristic CD spectra with negative peaks at 208 and 222 nm .
-
Thioflavin T (ThT) Binding Assay: This assay can be used to monitor potential β-sheet formation or aggregation, which would indicate misfolding. Correctly folded c subunit should show minimal ThT binding in its native state, while increased binding would suggest conformational changes toward amyloidogenic structures .
-
Reconstitution Assays: The functional verification of the c subunit can be assessed through reconstitution into liposomes or nanodiscs, followed by monitoring proton translocation activity or its ability to form oligomeric c-rings.
-
Protein-Protein Interaction Studies: Pull-down assays or co-immunoprecipitation with other ATP synthase subunits can verify the ability of the recombinant protein to maintain proper interactions within the complex.
It's worth noting that conformational changes can be induced by calcium, which promotes β-sheet formation and oligomerization. These calcium-dependent structural transitions can be monitored using the methods described above and provide valuable information about the protein's structural dynamics .
How does the structure and function of Guillardia theta ATP synthase subunit c compare to homologs in other organisms?
The ATP synthase subunit c shows remarkable evolutionary conservation across diverse organisms, from bacteria to complex eukaryotes including Guillardia theta. While maintaining the core functional domain, there are notable structural adaptations that reflect the specific physiological demands of different organisms.
Comparative analysis reveals:
| Organism Type | c-ring Stoichiometry | Key Structural Features | Functional Implications |
|---|---|---|---|
| Guillardia theta (Cryptophyte) | 10-14 subunits | α-helical hairpin, 81 amino acids | Optimized for chloroplast environment |
| Bacteria | 8-15 subunits | Simpler structure, fewer amino acids | Adapted to varied environmental conditions |
| Mitochondria | 8 subunits (typically) | Contains additional regulatory elements | Specialized for oxidative phosphorylation |
| Other Photosynthetic Eukaryotes | 10-14 subunits | Similar to G. theta but with species-specific adaptations | Optimized for specific photosynthetic requirements |
Research methodologies for comparative structural analysis typically involve homology modeling, multiple sequence alignments, and when possible, cryo-electron microscopy or X-ray crystallography to visualize the oligomeric arrangements of the c-ring.
What role does calcium play in modulating the structure and function of ATP synthase subunit c?
Calcium plays a significant and complex role in modulating ATP synthase subunit c structure and function, particularly regarding conformational changes and potential pathological implications. Research has shown that the c subunit is an amyloidogenic peptide that can undergo calcium-dependent conformational transitions.
This phenomenon can be studied using:
-
Fluorescence spectroscopy with amyloid-binding dyes such as Thioflavin T (ThT), which shows increased fluorescence upon binding to β-sheet structures.
-
Atomic force microscopy to visualize the fibril and oligomer formation.
-
Black lipid membrane methods to assess the functional consequences of these structural changes, particularly regarding membrane permeabilization.
The calcium-dependent conformational changes of ATP synthase subunit c may have significant implications for mitochondrial function during stress conditions. The c subunit has been proposed as a key participant in stress-induced inner mitochondrial membrane permeabilization by the mechanism of calcium-induced permeability transition. This pathological mechanism may share similarities with processes involving other amyloidogenic proteins like Aβ and α-synuclein .
For researchers studying Guillardia theta ATP synthase subunit c, understanding these calcium-dependent properties is crucial for interpreting experimental results and may provide insights into both physiological function and potential stress responses in chloroplasts.
What advanced techniques can be employed to study the assembly and dynamics of the c-ring in Guillardia theta ATP synthase?
Understanding the assembly and dynamics of the c-ring in Guillardia theta ATP synthase requires sophisticated methodological approaches that can capture both structural and functional aspects of this complex:
-
Cryo-Electron Microscopy (Cryo-EM): This technique has revolutionized our understanding of membrane protein complexes. For the c-ring, Cryo-EM can reveal the stoichiometry, arrangement, and interactions within the native complex without the need for crystallization. Sample preparation typically involves purification of the intact ATP synthase complex followed by vitrification and imaging.
-
Single-Molecule Fluorescence Resonance Energy Transfer (smFRET): By strategically labeling specific residues on the c subunit with fluorophore pairs, researchers can monitor conformational changes and rotational dynamics in real-time. This approach provides insights into the mechanism of proton translocation and c-ring rotation.
-
Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This technique can map the solvent accessibility of different regions of the protein, providing information about structural dynamics and protein-protein interactions within the complex.
-
Native Mass Spectrometry: For studying the intact c-ring assembly, native MS can determine the precise stoichiometry and stability of the complex under various conditions.
-
Molecular Dynamics Simulations: Computational approaches can model the behavior of the c-ring within the membrane environment, predicting conformational changes and proton translocation pathways that may be difficult to observe experimentally.
These techniques can be complemented by genetic approaches to study c-ring assembly. As seen in studies with Chlamydomonas reinhardtii, mutations affecting peripheral stalk subunits can impact ATP synthase biogenesis, suggesting coordinated assembly of the complex components . Similar approaches could be applied to Guillardia theta to understand the specific factors influencing c-ring assembly in this organism.
How can site-directed mutagenesis of recombinant Guillardia theta ATP synthase subunit c inform structure-function relationships?
Site-directed mutagenesis represents a powerful approach for investigating structure-function relationships in Guillardia theta ATP synthase subunit c. This methodology enables researchers to systematically alter specific amino acid residues and observe the resulting effects on protein structure, assembly, and function.
Key targets for mutagenesis studies include:
-
Proton-binding sites: Mutation of conserved acidic residues involved in proton binding and translocation can provide insights into the proton pathway and the mechanism of rotary catalysis.
-
Helix-helix interaction residues: Mutations at the interface between adjacent c subunits can reveal determinants of c-ring assembly and stability.
-
Lipid-interacting residues: Altering residues that interact with membrane lipids can help understand how the protein-lipid interface affects function.
-
Calcium-binding sites: Given the calcium-dependent conformational changes observed in ATP synthase subunit c , mutations in potential calcium-binding regions can elucidate the mechanism of calcium-induced structural transitions.
A comprehensive mutagenesis approach would involve:
| Mutation Type | Target Residues | Expected Outcomes | Analytical Methods |
|---|---|---|---|
| Conservative | Substitution with chemically similar amino acids | Minimal effect on structure, potential functional changes | Enzymatic activity assays, structural analysis |
| Non-conservative | Substitution with chemically distinct amino acids | Significant changes in structure and function | CD spectroscopy, functional reconstitution |
| Deletion | Removal of specific residues | Disruption of local structure or interactions | Assembly analysis, ThT binding |
| Insertion | Addition of residues at specific locations | Altered spacing or orientation | c-ring formation analysis |
Following mutagenesis, the mutant proteins must be characterized using the techniques described earlier (CD spectroscopy, ThT binding, reconstitution assays) to assess the impact on structure and function. Additionally, the ability of mutant proteins to form functional c-rings and integrate into the ATP synthase complex should be evaluated.
These experiments can provide valuable insights into how the primary sequence determines the structural features essential for proton translocation and ATP synthesis, as well as the molecular basis for calcium-induced conformational changes that may have implications for stress responses.
What are the challenges in studying the interaction between Guillardia theta ATP synthase subunit c and other subunits of the ATP synthase complex?
Investigating interactions between Guillardia theta ATP synthase subunit c and other components of the complex presents several significant challenges that require specialized methodological approaches:
To address these challenges, researchers can employ:
-
Crosslinking Mass Spectrometry (XL-MS): This approach uses chemical crosslinkers to capture protein-protein interactions followed by mass spectrometry identification of the crosslinked peptides. Zero-length crosslinkers or those with short spacer arms are particularly valuable for mapping precise interaction interfaces.
-
Förster Resonance Energy Transfer (FRET): By labeling subunit c and potential interaction partners with appropriate fluorophores, researchers can monitor interactions in real-time and under various conditions (e.g., different ATP concentrations, presence of inhibitors).
-
Nanodiscs or Styrene Maleic Acid Lipid Particles (SMALPs): These technologies provide more native-like membrane environments than detergent micelles, potentially preserving physiologically relevant interactions for structural and functional studies.
-
Co-evolution analysis: Computational approaches analyzing patterns of co-evolution between subunit c and other ATP synthase components can predict interaction interfaces, guiding experimental designs.
Each approach has specific technical considerations, and combining multiple complementary methods is often necessary to build a comprehensive understanding of the interaction network within the ATP synthase complex.
How can computational modeling enhance our understanding of Guillardia theta ATP synthase subunit c function?
Computational modeling offers powerful tools for investigating aspects of Guillardia theta ATP synthase subunit c function that may be challenging to study experimentally. These approaches provide atomic-level insights into dynamics, energetics, and mechanisms that complement experimental data.
Key computational approaches include:
-
Homology Modeling and Molecular Dynamics (MD) Simulations: Starting with the amino acid sequence of Guillardia theta ATP synthase subunit c, researchers can build structural models based on homologous proteins with known structures. These models can then be subjected to MD simulations to study:
-
Conformational dynamics of the c-ring
-
Proton binding and translocation pathways
-
Effects of membrane composition on protein behavior
-
Calcium binding and its effects on protein structure
-
-
Quantum Mechanics/Molecular Mechanics (QM/MM) Simulations: For detailed understanding of proton transfer mechanisms, QM/MM approaches can model the electronic processes involved in protonation/deprotonation events while considering the protein environment.
-
Coarse-Grained Simulations: These allow modeling of larger systems and longer timescales, enabling study of processes like c-ring assembly or interactions with other ATP synthase components.
-
Network Analysis and Machine Learning: By analyzing the patterns of amino acid conservation, co-evolution, and physical interactions, researchers can identify functionally important residues and predict the effects of mutations.
The methodological workflow typically involves:
| Computational Approach | Input Data | Output Information | Validation Method |
|---|---|---|---|
| Homology Modeling | Sequence, template structures | 3D structural model | RMSD to templates, energy minimization |
| MD Simulations | 3D structure, force field parameters | Dynamic behavior, energetics | Comparison with experimental observables |
| QM/MM | Selected regions for quantum treatment | Electronic properties, reaction energetics | Comparison with measured pKa values |
| Coarse-Grained Models | Simplified representation of protein | Large-scale dynamics, assembly | Comparison with cryo-EM data |
These computational approaches are particularly valuable for studying alternative conformational states that may be difficult to capture experimentally, such as the calcium-induced β-sheet structures or transient states during proton translocation. The predictions generated by these models can guide experimental design and provide mechanistic interpretations of experimental observations.
What techniques are most effective for studying the amyloidogenic properties of ATP synthase subunit c in research settings?
The amyloidogenic properties of ATP synthase subunit c represent an intriguing aspect of this protein that requires specialized techniques for comprehensive characterization. Recent research has demonstrated that the c subunit can form β-sheets and self-assemble into fibrils and oligomers in a calcium-dependent manner . The following methodological approaches are most effective for investigating these properties:
-
Thioflavin T (ThT) Fluorescence Assays: ThT is an amyloid-specific dye that exhibits enhanced fluorescence upon binding to cross-β structures. This technique allows real-time monitoring of amyloid formation kinetics under various conditions (pH, temperature, calcium concentration). The methodology involves:
-
Atomic Force Microscopy (AFM): AFM provides high-resolution imaging of amyloid structures, revealing morphological details of fibrils and oligomers. Sample preparation typically involves:
-
Transmission Electron Microscopy (TEM) with Negative Staining: This technique complements AFM by providing additional structural details of amyloid assemblies.
-
Circular Dichroism (CD) Spectroscopy: CD allows quantitative assessment of secondary structure changes associated with amyloid formation:
-
The transition from α-helical (native) to β-sheet (amyloid) structure is characterized by spectral shifts
-
Far-UV CD (190-260 nm) provides information on secondary structure content
-
Time-resolved CD can monitor structural transitions during amyloid formation
-
-
Black Lipid Membrane (BLM) Electrophysiology: This technique can assess the functional consequences of amyloid formation, particularly regarding membrane permeabilization:
For studying calcium dependence specifically, researchers should implement calcium titration experiments with each of these techniques, maintaining precise control over free calcium concentrations using appropriate buffers (e.g., EGTA/calcium mixtures). Additionally, site-directed mutagenesis targeting potential calcium-binding sites can help elucidate the molecular basis of calcium-induced amyloid formation.
How can researchers effectively distinguish between the physiological and pathological roles of ATP synthase subunit c?
Distinguishing between the physiological and potential pathological roles of ATP synthase subunit c requires methodological approaches that can selectively probe different structural states and functions of the protein. This distinction is particularly important given the evidence that the c subunit can adopt amyloidogenic conformations under certain conditions .
Effective research strategies include:
-
Structure-specific probes and assays:
-
Conformation-specific antibodies that recognize either the native α-helical or the alternative β-sheet conformations
-
Site-specific spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy to monitor local structural changes
-
Native gel electrophoresis to distinguish between properly assembled c-rings and abnormal oligomeric forms
-
-
Functional differentiation approaches:
-
Cellular and organellar systems:
-
Fluorescently-tagged c subunit variants to track localization and aggregation in vivo
-
Mitochondrial or chloroplast isolation followed by assessment of membrane integrity and function
-
Calcium challenge experiments to induce potential pathological transitions under controlled conditions
-
-
Comparative studies across species and conditions:
-
Parallel analysis of c subunits from different organisms to identify conserved physiological features versus species-specific pathological propensities
-
Systematic variation of environmental conditions (pH, ionic strength, lipid composition) to define the boundaries between physiological and pathological behaviors
-
| Experimental Approach | Physiological Function Indicator | Pathological Function Indicator |
|---|---|---|
| Structural Analysis | α-helical hairpin, defined c-ring | β-sheet formation, amorphous aggregates |
| ThT Binding | Minimal binding | Strong fluorescence increase |
| Membrane Effects | Proton translocation activity | Non-specific membrane permeabilization |
| Calcium Response | Regulated activity | Induced structural conversion and aggregation |
By combining these approaches, researchers can develop a comprehensive understanding of the conditions and mechanisms that govern the transition between the physiological role of ATP synthase subunit c in energy transduction and its potential involvement in pathological processes under stress conditions or disease states.
What are the emerging technologies that could advance the study of recombinant Guillardia theta ATP synthase subunit c?
Several cutting-edge technologies are poised to transform our understanding of Guillardia theta ATP synthase subunit c structure, function, and dynamics:
-
Cryo-Electron Tomography (Cryo-ET): This technique enables visualization of macromolecular complexes in their native cellular environment without the need for purification. For ATP synthase subunit c research, Cryo-ET could reveal:
-
Native arrangement of ATP synthase complexes in thylakoid membranes
-
Supramolecular organization and potential interactions with other complexes
-
Structural variations that might exist in vivo but be lost during purification
-
-
Single-Particle Cryo-EM with Improved Detectors: Recent advances in detector technology and image processing algorithms now enable atomic or near-atomic resolution of membrane protein complexes. Applied to Guillardia theta ATP synthase, this could resolve:
-
Precise c-ring stoichiometry and arrangement
-
Detailed interaction interfaces between subunit c and other components
-
Conformational states relevant to the catalytic cycle
-
-
Advanced Spectroscopic Techniques:
-
Solid-state NMR methods optimized for membrane proteins could provide atomic-level insights into c subunit dynamics
-
Time-resolved serial crystallography at X-ray free-electron lasers (XFELs) could capture transient states during proton translocation
-
-
Genome Editing Technologies:
-
CRISPR-Cas9 systems adapted for Guillardia theta could enable precise genetic manipulation
-
Creation of tagged variants for in vivo tracking and functional studies
-
Generation of conditional mutants to study essential functions
-
-
Artificial Intelligence and Machine Learning:
-
Improved prediction of protein-protein interactions and complex assembly
-
Enhanced computational models of c-ring dynamics and proton translocation
-
Automated analysis of structural data from multiple experimental sources
-
These technologies, particularly when used in combination, have the potential to address longstanding questions about ATP synthase function and regulation in cryptophytes, potentially revealing unique adaptations in Guillardia theta compared to other photosynthetic organisms.
How might understanding Guillardia theta ATP synthase subunit c contribute to bioenergetics research and applications?
Research on Guillardia theta ATP synthase subunit c has significant potential to advance fundamental bioenergetics understanding and inspire novel applications across several domains:
-
Evolutionary Insights into Energy Conservation:
-
Cryptophytes like Guillardia theta represent a unique evolutionary lineage with a complex history of endosymbiosis
-
Comparative analysis of ATP synthase components across diverse photosynthetic organisms can illuminate the evolution of energy conversion mechanisms
-
Understanding the specific adaptations in Guillardia theta ATP synthase could reveal alternative solutions to the universal challenge of energy conservation
-
-
Biomimetic Energy Conversion Systems:
-
The natural rotary mechanism of ATP synthase represents one of the most efficient energy conversion systems known
-
Detailed structural and functional characterization of Guillardia theta ATP synthase components could inform the design of synthetic nanomotors or molecular machines
-
The specific properties of the c-ring, including its proton translocation mechanism, could inspire artificial proton-conducting systems
-
-
Biotechnological Applications:
-
Therapeutic Relevance:
-
The amyloidogenic properties of ATP synthase subunit c may have parallels with other proteins involved in neurodegenerative diseases
-
Understanding the mechanisms controlling conformational transitions between α-helical and β-sheet structures could provide insights relevant to disorders involving protein misfolding
-
Potential development of strategies to prevent pathological transitions while preserving essential functions
-
-
Sustainable Energy Research:
-
Insights from biological energy conversion systems like ATP synthase could inform the development of more efficient artificial photosynthetic systems
-
Understanding how Guillardia theta maintains ATP synthase efficiency under variable environmental conditions could suggest strategies for robust bioenergy applications
-
The methodological approaches required for these investigations include interdisciplinary techniques spanning structural biology, biochemistry, biophysics, synthetic biology, and materials science. Collaborative research bridging these disciplines will be essential to translate fundamental insights about Guillardia theta ATP synthase into impactful applications.
