Recombinant Psilotum nudum ATP synthase subunit c, chloroplastic (atpH)

What is the function of ATP synthase subunit c in chloroplasts?

ATP synthase subunit c in chloroplasts is a critical component of the ATP synthase complex, which produces adenosine triphosphate (ATP) required for photosynthetic metabolism. The c-subunit forms an oligomeric ring (c₍ₙ₎) embedded in the thylakoid membrane. The synthesis of ATP is mechanically coupled to the rotation of this c-subunit ring, which is driven by the translocation of protons across the membrane along an electrochemical gradient. This proton movement through the c-ring powers the conformational changes in the F₁ portion of the enzyme, enabling ATP synthesis .

The c-subunit specifically functions as part of the membrane-embedded F₀ sector of the ATP synthase complex, forming the pathway for protons to move across the membrane. This proton translocation is essential for converting the electrochemical potential energy stored in the proton gradient into the mechanical energy of rotation that drives ATP synthesis .

How does Psilotum nudum ATP synthase subunit c compare to homologous proteins in other organisms?

Psilotum nudum ATP synthase subunit c shares significant homology with other chloroplastic ATP synthase c subunits, particularly those from early-diverging plant lineages. Comparative genomic analyses show that P. nudum, as a primitive vascular plant (whisk fern), possesses a chloroplast ATP synthase with features that provide evolutionary insights into the development of photosynthetic machinery .

The P. nudum ATP synthase subunit c shows closer sequence similarity to those found in other non-flowering plants like ferns (e.g., Adiantum capillus-veneris) compared to angiosperms. Additionally, phylogenetic analyses indicate that chloroplast ATP synthase components in plants like P. nudum show stronger homology to cyanobacterial ATP synthases than to those found in other bacteria or mitochondria, supporting the endosymbiotic theory of chloroplast evolution .

Comparative studies reveal that while the core function of ATP synthase subunit c is conserved across photosynthetic organisms, the stoichiometry of c-subunits in the c-ring (c₍ₙ₎) varies between species, which affects the bioenergetic efficiency of ATP synthesis by altering the proton-to-ATP ratio .

What are the optimal expression systems for recombinant production of Psilotum nudum ATP synthase subunit c?

The optimal expression system for recombinant production of Psilotum nudum ATP synthase subunit c involves a bacterial expression system with specific modifications to accommodate the hydrophobic nature of this membrane protein. Based on successful approaches with similar chloroplastic c-subunits, the following methodology is recommended:

• Expression vector selection: Use a bacterial expression vector containing a fusion tag partner like maltose binding protein (MBP) to enhance solubility. This approach has been successfully employed for spinach chloroplast ATP synthase c1 subunit .

• Host strain optimization: Utilize BL21 derivative Escherichia coli cells, which have proven effective for the expression of eukaryotic membrane proteins including chloroplastic ATP synthase components .

• Codon optimization: Implement codon optimization of the atpH gene insert in the expression plasmid to enhance translation efficiency in the bacterial host, as the codon usage in P. nudum chloroplast genes may differ significantly from that of E. coli .

• Expression conditions: Culture bacterial cells at lower temperatures (16-20°C) after induction to slow protein synthesis and allow proper folding of the membrane protein, with induction using lower IPTG concentrations (0.1-0.5 mM) to prevent formation of inclusion bodies.

• Solubilization approach: Express the hydrophobic c-subunit as a soluble fusion protein (e.g., MBP-c1) to overcome the aggregation issues typically associated with membrane protein expression .

What purification methods are most effective for recombinant Psilotum nudum ATP synthase subunit c?

The most effective purification strategy for recombinant Psilotum nudum ATP synthase subunit c involves a multi-step approach designed to address the hydrophobic nature of this membrane protein:

• Initial affinity chromatography: Utilize affinity chromatography targeting the fusion tag (e.g., MBP) to capture the fusion protein from bacterial lysate. A typical approach uses amylose resin for MBP-tagged proteins with elution using maltose-containing buffer .

• Proteolytic cleavage: Implement site-specific protease treatment (e.g., TEV protease or Factor Xa) to cleave the c-subunit from its fusion partner under controlled conditions that minimize aggregation .

• Reversed-phase chromatography: Apply the cleaved protein mixture to a reversed-phase column for final purification. This approach has been successfully used for spinach chloroplast ATP synthase c1 subunit and works well for hydrophobic membrane proteins .

• Storage considerations: Store the purified protein in an appropriate buffer (Tris-based buffer with 50% glycerol) at -20°C for short-term storage or -80°C for extended storage. Avoid repeated freeze-thaw cycles as they can compromise protein integrity .

• Quality control: Confirm the structural integrity of the purified c-subunit using circular dichroism spectroscopy to verify the correct alpha-helical secondary structure, which is essential for functional studies .

How can researchers verify the correct folding and functionality of recombinant Psilotum nudum ATP synthase subunit c?

Verifying the correct folding and functionality of recombinant Psilotum nudum ATP synthase subunit c requires multiple analytical approaches:

• Secondary structure analysis: Employ circular dichroism (CD) spectroscopy to confirm the alpha-helical secondary structure characteristic of properly folded c-subunits. The CD spectrum should show typical negative peaks at 208 and 222 nm consistent with alpha-helical content .

• Size exclusion chromatography: Assess the oligomeric state and homogeneity of the protein preparation using size exclusion chromatography, which can distinguish between monomeric forms and potential aggregates.

• Reconstitution studies: Reconstitute the purified c-subunit into liposomes or nanodiscs to assess membrane integration and potential for oligomeric ring formation. This approach can be used to evaluate the capacity of the recombinant protein to form functional structures.

• Proton translocation assays: Measure proton translocation capabilities in reconstituted systems using pH-sensitive fluorescent dyes (e.g., ACMA or pyranine) to assess functionality.

• Structural integration tests: Evaluate the ability of the recombinant c-subunit to assemble with other ATP synthase components in reconstitution experiments, which would indicate proper folding and functional capacity.

How can recombinant Psilotum nudum ATP synthase subunit c be used to study c-ring stoichiometry variation across species?

Recombinant Psilotum nudum ATP synthase subunit c provides a valuable tool for investigating the evolutionary and functional significance of c-ring stoichiometry variation across species. The following methodological approaches can be employed:

• Reconstitution experiments: Combine purified recombinant P. nudum c-subunits under controlled conditions to study their self-assembly properties. Variables such as lipid composition, pH, and ionic strength can be manipulated to examine factors influencing c-ring formation and stoichiometry .

• Cross-linking studies: Implement chemical cross-linking followed by mass spectrometry analysis to determine the number of c-subunits in reconstituted rings, providing direct evidence of stoichiometry.

• Hybrid ring formation: Mix recombinant c-subunits from P. nudum with those from other species to investigate compatibility and preferences in oligomerization, revealing evolutionary constraints on c-ring assembly.

• Structure-function analysis: Introduce site-directed mutations in the recombinant protein to identify specific amino acid residues that influence c-ring formation and stability, thereby elucidating the molecular basis of stoichiometric variation .

• Bioenergetic studies: Correlate c-ring stoichiometry with bioenergetic parameters such as proton-to-ATP ratios in different photosynthetic organisms, which can provide insights into the adaptive significance of stoichiometric variation in relation to environmental niches .

What are the challenges in crystallization of Psilotum nudum ATP synthase subunit c for structural studies?

Crystallization of Psilotum nudum ATP synthase subunit c presents several significant challenges that researchers must address:

• Hydrophobicity management: The highly hydrophobic nature of the c-subunit makes it prone to aggregation in aqueous solutions, requiring specialized approaches such as the use of detergents, lipidic cubic phases, or amphipols to maintain solubility during crystallization trials.

• Conformational homogeneity: Ensuring a homogeneous population of properly folded protein is crucial for successful crystallization. This can be particularly challenging for membrane proteins like the c-subunit, which may adopt multiple conformations depending on the surrounding environment.

• Crystal packing considerations: The cylindrical shape of the c-ring assembly and its hydrophobic surfaces can hinder the formation of ordered crystal lattices. Approaches such as co-crystallization with antibody fragments or designing specialized crystallization chaperones may be necessary.

• Detergent selection: The choice of detergent is critical for maintaining the native structure of the protein while allowing crystal contacts to form. Systematic screening of various detergents and detergent concentrations is typically required.

• Alternative structural approaches: When crystallization proves challenging, researchers may consider alternative structural biology techniques such as cryo-electron microscopy, which has become increasingly powerful for membrane protein structure determination, or solid-state NMR spectroscopy.

How can researchers investigate the interaction between Psilotum nudum ATP synthase subunit c and other components of the ATP synthase complex?

Investigating interactions between Psilotum nudum ATP synthase subunit c and other components of the ATP synthase complex requires sophisticated biochemical and biophysical approaches:

• Co-immunoprecipitation studies: Utilize antibodies against the c-subunit or other ATP synthase components to pull down interacting partners from solubilized membrane preparations, followed by identification using mass spectrometry.

• Cross-linking mass spectrometry (XL-MS): Apply chemical cross-linking reagents to capture transient or stable interactions between the c-subunit and other ATP synthase components, followed by proteolytic digestion and mass spectrometric analysis to identify cross-linked peptides that reveal interaction sites.

• Surface plasmon resonance (SPR): Immobilize purified recombinant c-subunit on a sensor chip and measure binding kinetics with other purified ATP synthase components to quantify interaction strengths and dynamics.

• Förster resonance energy transfer (FRET): Label the c-subunit and potential interaction partners with compatible fluorophores to monitor their proximity and interaction in reconstituted systems or native membranes.

• Molecular docking and dynamics simulations: Combine structural data with computational approaches to model interactions between the c-subunit and other components, generating testable hypotheses about key residues involved in these interactions.

How does the structure of Psilotum nudum ATP synthase subunit c reflect its evolutionary position among photosynthetic organisms?

The structure of Psilotum nudum ATP synthase subunit c provides valuable insights into the evolutionary history of photosynthetic machinery:

• Phylogenetic context: As a primitive vascular plant (whisk fern), P. nudum occupies an important evolutionary position, representing an early diverging lineage of vascular plants. Its ATP synthase subunit c structure reflects this transitional position between non-vascular plants and more derived vascular plants .

• Cyanobacterial relationship: The P. nudum ATP synthase subunit c shows stronger sequence homology to cyanobacterial homologues than to those from other bacteria or mitochondria, supporting the endosymbiotic origin of chloroplasts. This relationship is evident in both sequence conservation and structural features .

• Conserved functional domains: Despite evolutionary divergence, the P. nudum c-subunit maintains highly conserved functional domains involved in proton translocation and c-ring formation, demonstrating the fundamental importance of these features for ATP synthesis across the evolutionary spectrum .

• Unique adaptations: Comparative analysis reveals specific amino acid substitutions in the P. nudum c-subunit that may represent adaptations to the specific physiological conditions and energetic requirements of this early vascular plant lineage.

• Stoichiometric implications: The sequence characteristics of P. nudum ATP synthase subunit c may influence the stoichiometry of the c-ring, potentially reflecting evolutionary adaptations in the bioenergetic efficiency of ATP synthesis in response to ecological factors .

What insights can be gained from comparing Psilotum nudum ATP synthase genes with those in other plant chloroplasts?

Comparative analysis of Psilotum nudum ATP synthase genes with those in other plant chloroplasts yields significant evolutionary and functional insights:

• Gene organization: The organization of ATP synthase genes in the P. nudum chloroplast genome shows similarities to that observed in other non-flowering plants, particularly ferns like Adiantum capillus-veneris, while differing from the arrangements seen in angiosperms. This provides evidence for the evolutionary trajectory of chloroplast genome organization .

• Codon usage patterns: Analysis of codon usage in the atpH gene of P. nudum reveals patterns that reflect its evolutionary position and may influence translation efficiency in the chloroplast environment. These patterns can be compared with those in other plant lineages to understand evolutionary pressures on chloroplast gene expression.

• Regulatory elements: Comparison of promoter regions and other regulatory elements associated with ATP synthase genes across plant lineages can reveal the evolution of expression control mechanisms in the chloroplast.

• Evolutionary rate: The rate of sequence evolution in P. nudum ATP synthase genes compared to those in other plant groups provides insights into the selective pressures acting on these essential components of the photosynthetic apparatus.

• Gene transfer events: Evidence for potential gene transfer events between the chloroplast and nuclear genomes can be assessed through comparative analysis, contributing to our understanding of the ongoing evolutionary dynamics between these genomic compartments.

How do the functional characteristics of Psilotum nudum ATP synthase subunit c compare with bacterial and mitochondrial counterparts?

The functional characteristics of Psilotum nudum ATP synthase subunit c reveal important distinctions when compared to bacterial and mitochondrial counterparts:

• Sequence relationships: Chloroplastic ATP synthase subunit c from P. nudum shows closer sequence homology to cyanobacterial homologues than to those from other bacteria or mitochondria, reflecting the endosymbiotic origin of chloroplasts from cyanobacterial ancestors .

• Structural adaptations: While the core structure of ATP synthase subunit c is conserved across domains of life, the P. nudum protein displays specific adaptations to the chloroplast environment, particularly in regions that interact with other ATP synthase components and the lipid bilayer.

• Proton-binding site: The critical glutamate residue involved in proton binding and translocation is conserved across bacterial, chloroplastic, and mitochondrial c-subunits, highlighting the fundamental conservation of the proton translocation mechanism .

• C-ring stoichiometry: The number of c-subunits in the c-ring varies between species and organelles, affecting the bioenergetic efficiency of ATP synthesis. In chloroplasts like those of P. nudum, the c-ring typically contains 14 subunits, compared to 8-15 in bacteria and 8 in mammalian mitochondria, reflecting adaptations to different bioenergetic environments .

• Interaction with other ATP synthase components: The P. nudum c-subunit has evolved specific interaction surfaces for chloroplast-specific ATP synthase components, distinguishing it functionally from bacterial and mitochondrial counterparts despite their common evolutionary origin .

What are the most common obstacles in expressing and purifying recombinant Psilotum nudum ATP synthase subunit c?

Researchers face several technical challenges when working with recombinant Psilotum nudum ATP synthase subunit c:

• Protein aggregation: The hydrophobic nature of the c-subunit leads to aggregation during expression and purification. This can be addressed by expressing the protein as a fusion with solubility-enhancing partners like MBP, and using appropriate detergents during purification .

• Low expression yields: Membrane proteins often express at lower levels than soluble proteins. Optimization strategies include using specialized expression strains, adjusting induction conditions (temperature, inducer concentration, induction time), and implementing codon optimization for the expression host .

• Protein misfolding: Ensuring proper folding of the recombinant protein is critical. Solutions include expression at lower temperatures, co-expression with molecular chaperones, and careful selection of solubilization and purification conditions that maintain native-like environments .

• Proteolytic degradation: The recombinant protein may be susceptible to proteolysis during expression and purification. This can be mitigated by using protease-deficient host strains, including protease inhibitors during purification, and optimizing purification protocols to minimize processing time.

• Storage stability: Maintaining the stability of the purified protein during storage is challenging. Recommended approaches include storage in a Tris-based buffer with 50% glycerol at -20°C or -80°C, avoiding repeated freeze-thaw cycles, and maintaining aliquots at 4°C for short-term use .

How can researchers overcome the challenges in studying membrane proteins like ATP synthase subunit c?

Studying membrane proteins like ATP synthase subunit c presents unique challenges that require specialized approaches:

• Solubilization strategies: Select appropriate detergents or amphipathic polymers that effectively solubilize the membrane protein while preserving its native structure. For ATP synthase subunit c, detergents like n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) are often effective .

• Membrane mimetic systems: Utilize membrane mimetic systems such as nanodiscs, liposomes, or bicelles to provide a native-like environment for functional and structural studies of the c-subunit after purification.

• Advanced expression systems: Consider cell-free expression systems specifically designed for membrane proteins, which allow direct incorporation into provided lipid environments during synthesis.

• Structural biology approaches: Implement specialized structural biology techniques suitable for membrane proteins, such as cryo-electron microscopy, electron crystallography, or solid-state NMR, which can overcome limitations of traditional X-ray crystallography .

• Computational predictions: Complement experimental approaches with computational methods such as molecular dynamics simulations to predict membrane protein behavior and interactions within the lipid bilayer.

What quality control methods ensure the integrity of recombinant Psilotum nudum ATP synthase subunit c preparations?

Ensuring the quality and integrity of recombinant Psilotum nudum ATP synthase subunit c preparations requires rigorous assessment through multiple complementary methods:

• SDS-PAGE and western blotting: Use these techniques to verify protein purity, molecular weight, and immunoreactivity. For membrane proteins like ATP synthase subunit c, specialized SDS-PAGE systems designed for hydrophobic proteins may be necessary.

• Circular dichroism spectroscopy: Confirm the correct secondary structure of the purified protein, which for ATP synthase subunit c should show predominantly alpha-helical characteristics with negative peaks at 208 and 222 nm in the CD spectrum .

• Mass spectrometry: Verify the exact mass and sequence integrity of the purified protein using techniques such as MALDI-TOF or ESI-MS, which can also identify any post-translational modifications or degradation products.

• Dynamic light scattering: Assess the homogeneity and oligomeric state of the protein preparation, which can reveal potential aggregation issues or the formation of appropriate oligomeric structures.

• Functional assays: Test the functional integrity of the purified protein through reconstitution experiments that assess its ability to form c-rings and participate in proton translocation, providing the ultimate validation of proper folding and structural integrity.

What emerging technologies could advance our understanding of Psilotum nudum ATP synthase subunit c?

Several cutting-edge technologies hold promise for advancing research on Psilotum nudum ATP synthase subunit c:

• Cryo-electron microscopy (cryo-EM): The recent "resolution revolution" in cryo-EM makes it possible to determine high-resolution structures of membrane protein complexes like ATP synthase without the need for crystallization, potentially revealing the detailed arrangement of c-subunits in the native context of the complete ATP synthase complex.

• Native mass spectrometry: Advanced native MS approaches can determine the precise stoichiometry and composition of intact membrane protein complexes, providing direct evidence of c-ring composition and interactions with other ATP synthase components.

• Single-molecule biophysics: Techniques such as single-molecule FRET and high-speed atomic force microscopy can directly visualize the dynamics of individual ATP synthase complexes, potentially capturing the rotational movement of the c-ring during ATP synthesis.

• In-cell structural biology: Emerging methods for structural studies within intact cells, such as in-cell NMR and cryo-electron tomography, could reveal the native conformation and interactions of ATP synthase components in their cellular context.

• Synthetic biology approaches: Designer ATP synthases with modified c-subunits could be engineered to test hypotheses about c-ring stoichiometry and function, potentially leading to novel biotechnological applications.

How can understanding Psilotum nudum ATP synthase subunit c contribute to broader research in bioenergetics?

Research on Psilotum nudum ATP synthase subunit c has significant implications for broader bioenergetic studies:

• Evolutionary bioenergetics: As a member of an early-diverging vascular plant lineage, P. nudum provides a valuable reference point for understanding the evolution of photosynthetic energy conversion machinery across plant lineages.

• Structure-function relationships: Detailed characterization of the P. nudum c-subunit structure and function can reveal fundamental principles governing the proton-driven rotary mechanism of ATP synthases across domains of life.

• Energy conversion efficiency: Studies on c-ring stoichiometry in P. nudum can illuminate how organisms optimize the balance between ATP synthesis rate and efficiency, providing insights into bioenergetic adaptations.

• Biomimetic applications: Understanding the molecular details of proton translocation through the c-ring could inspire the development of artificial proton conductors or synthetic energy conversion devices.

• Comparative bioenergetics: Systematic comparison of ATP synthase components across species can reveal convergent and divergent solutions to the fundamental challenge of energy conversion, contributing to our understanding of bioenergetic principles.

What interdisciplinary approaches could enhance research on chloroplastic ATP synthase components?

Interdisciplinary approaches offer powerful new avenues for investigating chloroplastic ATP synthase components like the P. nudum subunit c:

• Structural bioinformatics and artificial intelligence: Advanced computational methods, including AI-based structure prediction tools like AlphaFold2, can generate high-confidence structural models of ATP synthase components and their complexes, guiding experimental design.

• Systems biology: Integration of proteomics, transcriptomics, and metabolomics data can provide a comprehensive view of how ATP synthase function relates to broader cellular processes and metabolic networks in P. nudum and other photosynthetic organisms.

• Evolutionary developmental biology (evo-devo): Comparing ATP synthase structure and function across diverse photosynthetic lineages can reveal how this essential machinery has been conserved or modified during plant evolution.

• Synthetic biology and protein engineering: Rational design and directed evolution approaches can be applied to modify the properties of ATP synthase components, potentially creating variants with enhanced stability or altered functionality for biotechnological applications.

• Quantitative biophysics: Combining structural data with biophysical measurements and thermodynamic analysis can provide a more complete understanding of the energetics and dynamics of ATP synthesis, connecting molecular structure to function.