Recombinant Nicotiana tomentosiformis ATP synthase subunit c, chloroplastic (atpH)

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

ATP synthase subunit c (atpH) is a critical component of the chloroplastic ATP synthase complex, which produces the adenosine triphosphate (ATP) required for photosynthetic metabolism. The c-subunit forms a multimeric ring (c₁₀-c₁₅) embedded in the thylakoid membrane. This structure's rotation is mechanically coupled to ATP synthesis through a process driven by proton translocation across the membrane along an electrochemical gradient .

The c-subunit ring functions as part of the F₀ sector of ATP synthase. Its rotation is coupled to the γ-stalk in the F₁ region, where the γ-subunit acts as a shaft inside the α₃β₃ head. This rotational motion drives the catalysis of ADP + Pᵢ → ATP at the three α-β subunit interfaces in F₁ .

How does the c-subunit stoichiometry affect ATP synthase efficiency?

The stoichiometry of c-subunits in the ring varies among organisms, ranging from c₁₀ to c₁₅. This variation directly affects the coupling ratio (ions transported : ATP generated), which can range from 3.3 to 5.0 . While the number of ATP molecules generated per c-ring rotation remains constant at 3 across all known ATP synthases, the number of protons required for each complete rotation varies according to the number of c-subunits in the ring.

This stoichiometric variation has significant implications for ATP synthesis efficiency. A higher number of c-subunits requires more protons per ATP molecule produced, but may offer advantages under specific physiological or environmental conditions. Though various hypotheses have been proposed, the exact evolutionary and functional significance of this stoichiometric variation remains under investigation .

What expression systems are most effective for recombinant atpH production?

The most widely used and effective expression system for recombinant atpH production is Escherichia coli. Research demonstrates successful expression of the atpH protein from various plant species including Nicotiana tomentosiformis, Nicotiana sylvestris, Panax ginseng, and Spinacia oleracea in E. coli systems .

For optimal expression in E. coli, researchers have developed the following methodology:

• Gene design with codon optimization for E. coli expression

• Addition of terminal restriction sites for cloning

• Use of expression vectors that incorporate fusion tags (commonly His-tags) to facilitate purification

• Induction of protein expression using IPTG at concentrations around 1.0 mM

• Optimization of incubation times (typically 30 minutes post-induction)

This approach yields significant quantities of highly purified c₁ subunit while confirming that the purified protein maintains the correct alpha-helical secondary structure essential for function .

What are the optimal purification strategies for recombinant atpH protein?

Purification of recombinant atpH presents challenges due to its hydrophobic nature as a membrane protein. Based on current research, the following purification strategy has proven most effective:

• Cell lysis using a combination of:

  • Lysis buffer (20 mM Tris-HCl pH 8.0 with protease inhibitor cocktail)

  • Lysozyme treatment (1 mg/mL) with incubation at 4°C for 1.5 hours

  • Sonication at 50-75 W

• Affinity chromatography using the fusion tag (typically His-tag) for initial purification

• Verification of purity using SDS-PAGE analysis, with >90% purity achievable through optimized protocols

• Storage in appropriate buffer conditions:

  • Tris/PBS-based buffer with 6% Trehalose, pH 8.0

  • Addition of 5-50% glycerol for long-term storage

  • Storage at -20°C/-80°C to maintain stability

For researchers working with this protein, it's important to note that repeated freeze-thaw cycles should be avoided, and working aliquots should be maintained at 4°C for no more than one week .

How can recombinant atpH be used to investigate c-ring stoichiometry and assembly?

Recombinant atpH production enables several advanced research applications for investigating c-ring stoichiometry and assembly:

• Reconstitution studies: Purified monomeric c₁ subunits can be used in reconstitution experiments aimed at forming multimeric c-rings (c₍ₙ₎) in vitro. This approach allows researchers to study the factors affecting ring assembly and stoichiometry under controlled conditions .

• Site-directed mutagenesis: The recombinant expression system allows for precise genetic modifications to investigate how specific amino acid residues influence c-ring assembly, stability, and proton translocation efficiency .

• Comparative analyses: By producing recombinant atpH from different species, researchers can directly compare structural and functional characteristics to understand evolutionary adaptations and species-specific variations in c-ring stoichiometry .

• Hybrid ring formation: Combining wild-type and mutant c-subunits in varying ratios enables investigation of cooperative assembly processes and minimum structural requirements for functional ring formation.

These approaches provide valuable tools for addressing the undefined factors that affect c-ring stoichiometry and structure, potentially revealing new insights into ATP synthase evolution and adaptation .

What methods are available for studying the interaction between atpH and other ATP synthase components?

Several methodological approaches are available for investigating interactions between atpH and other ATP synthase components:

• Cross-linking studies: Chemical cross-linking combined with mass spectrometry can identify specific interaction sites between c-subunits and other components of the ATP synthase complex.

• Förster resonance energy transfer (FRET): By introducing fluorescent tags at strategic positions, researchers can measure distances and conformational changes between the c-ring and other subunits during rotation and ATP synthesis.

• Electron microscopy and cryo-EM: These techniques provide structural insights into the assembled ATP synthase complex, revealing the arrangement and interactions of the c-ring with other components at near-atomic resolution.

• Site-directed spin labeling: This approach allows for the investigation of dynamic interactions and conformational changes using electron paramagnetic resonance (EPR) spectroscopy.

• Biochemical binding assays: Using recombinant components, researchers can quantify binding affinities and kinetics between the c-subunit and other ATP synthase proteins.

How does temperature affect atpH function in chloroplast ATP synthase?

• Cold temperature exposure creates challenges for photosynthetic organisms by potentially causing electron imbalances and oxidative damage .

• Plants respond to cold conditions by upregulating production of Calvin Cycle enzymes, including components of ATP synthase, to maintain energy balance and photosynthetic efficiency .

• Chloroplast RNA-binding proteins like CP29A play essential roles in cold adaptation by regulating gene expression, including potentially affecting the expression of ATP synthase components .

• CRISPR/Cas9-induced mutations affecting chloroplast gene expression regulators have demonstrated cold-dependent photosynthetic deficiencies, highlighting the importance of proper regulation of chloroplast proteins like atpH under temperature stress .

For researchers investigating atpH function across temperature gradients, experimental designs should include:

• Controlled temperature treatments

• Measurement of ATP synthesis rates at different temperatures

• Analysis of c-ring stability and proton conductance under temperature stress

• Investigation of potential post-translational modifications that might regulate atpH function in response to temperature changes

What role does atpH play in plant adaptation to varying light conditions?

The ATP synthase c-subunit (atpH) plays a crucial role in plant adaptation to varying light conditions through its participation in photosynthetic energy conversion. Research findings indicate:

• The ATP synthase complex, including the c-ring, is part of a dynamic redox regulatory network that responds to light transitions. After a dark-to-light transition, redox regulators gradually reduce targets until a steady state is reached, with the rate dependent on light intensity .

• During induction of photosynthesis, the thylakoid membrane undergoes significant adjustments, including regulation of proton motive force that directly affects ATP synthase function .

• The chloroplast redox network involves multiple components including thioredoxins (TRXs), which can affect ATP synthase activity through redox regulation .

For researchers investigating atpH's role in light adaptation, the following experimental approaches are recommended:

• Analysis of ATP synthase activity during dark-to-light transitions

• Measurement of proton conductance through the c-ring under varying light intensities

• Investigation of potential redox modifications of the c-subunit

• Comparative studies of atpH sequence and function across plant species adapted to different light environments

What spectroscopic methods are most informative for analyzing recombinant atpH structure?

Several spectroscopic methods provide valuable information about the structure of recombinant atpH:

• Circular Dichroism (CD) Spectroscopy: This technique is particularly useful for confirming the alpha-helical secondary structure that is characteristic of the c-subunit. Research has confirmed that purified recombinant c₁ maintains the correct alpha-helical secondary structure, which is essential for proper function .

• Fourier Transform Infrared Spectroscopy (FTIR): Provides information about protein secondary structure in membrane environments, enabling analysis of how the c-subunit interacts with lipids.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: Can provide atomic-level structural information about the c-subunit, especially when isotopically labeled protein is produced.

• Mass Spectrometry: Useful for confirming protein identity, post-translational modifications, and interaction sites with other subunits through cross-linking studies.

For optimal results, researchers should consider combining multiple spectroscopic approaches to obtain comprehensive structural information about recombinant atpH in different environments.

How can molecular dynamics simulations complement experimental studies of atpH?

Molecular dynamics (MD) simulations offer powerful complementary approaches to experimental studies of atpH by providing insights that may be difficult to obtain through experimental methods alone:

• Membrane Integration Analysis: MD simulations can model how the c-subunit integrates into lipid bilayers, revealing key lipid-protein interactions that stabilize the c-ring in the thylakoid membrane.

• Proton Translocation Mechanisms: Simulations can elucidate the detailed molecular mechanisms of proton binding, translocation, and release as the c-ring rotates, including the identification of key residues involved in these processes.

• Ring Assembly Dynamics: MD approaches can model the assembly of individual c-subunits into the complete ring structure, identifying critical interactions that determine ring size and stability.

• Prediction of Mutation Effects: Before experimental validation, simulations can predict how specific mutations might affect c-ring structure, stability, and function.

• Environmental Response Modeling: MD simulations can predict how changes in temperature, pH, or ionic strength might affect c-ring dynamics and function.

For optimal results, MD simulation studies should be designed with parameters derived from experimental structural data and validated against experimental functional measurements.

How does Nicotiana tomentosiformis atpH compare structurally and functionally to atpH from other plant species?

Comparative analysis reveals important similarities and differences between Nicotiana tomentosiformis atpH and corresponding proteins from other plant species:

This remarkable conservation of the atpH protein sequence across diverse plant species suggests strong evolutionary constraints maintaining the structure and function of this critical ATP synthase component. Despite the evolutionary distance between tobacco plants and ginseng, the identical amino acid sequences indicate essential functional requirements that tolerate minimal variation .

Researchers investigating species-specific aspects of atpH should focus on:

• Potential differences in post-translational modifications

• Variations in gene expression regulation

• Possible differences in c-ring assembly and stoichiometry

• Environmental adaptations that might affect ATP synthase function without altering the primary sequence

What methodological approaches are most effective for studying species-specific variations in atpH function?

To effectively investigate species-specific variations in atpH function, researchers should employ multiple complementary methodologies:

• Comparative Genomics and Transcriptomics:

  • Analysis of atpH gene sequences across species

  • Examination of regulatory regions that might influence expression

  • Investigation of RNA processing and stability mechanisms

  • Quantification of gene expression under various environmental conditions

• Heterologous Expression Systems:

  • Production of atpH from different species in E. coli using standardized methods

  • Creation of chimeric proteins to identify functionally important regions

  • Development of in vitro reconstitution systems using components from different species

• Structural Comparison Techniques:

  • Cryo-electron microscopy of ATP synthase complexes from different species

  • Spectroscopic analysis of purified recombinant proteins

  • Computational modeling to identify subtle structural differences

• Functional Assays:

  • Measurement of ATP synthesis rates under various conditions

  • Proton translocation assays

  • Analysis of c-ring stability and assembly efficiency

  • Investigation of temperature and pH optima

• In vivo Complementation Studies:

  • Introduction of recombinant atpH from various species into model organisms

  • CRISPR/Cas9-mediated replacement of native atpH with variants from other species

  • Phenotypic analysis under various environmental stresses

These approaches, when used in combination, can reveal subtle species-specific adaptations in atpH function that might not be apparent from sequence analysis alone .

What are common challenges in recombinant atpH expression and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant atpH expression systems:

• Protein Toxicity in Expression Host:

  • Challenge: The hydrophobic nature of atpH can disrupt host cell membranes.

  • Solution: Use tightly regulated expression systems, lower induction temperatures (16-20°C), and shorter induction times (30 minutes has proven effective) .

• Protein Aggregation and Inclusion Body Formation:

  • Challenge: Membrane proteins often form insoluble aggregates.

  • Solution: Fusion with solubility-enhancing tags like maltose-binding protein (MBP), use of mild detergents during extraction, and optimization of cell lysis conditions .

• Low Expression Yields:

  • Challenge: Membrane proteins typically express at lower levels than soluble proteins.

  • Solution: Codon optimization for the expression host, use of specialized expression strains (C41/C43), and optimization of media composition and growth conditions .

• Purification Difficulties:

  • Challenge: Separating the target protein from host membrane proteins.

  • Solution: Use of affinity tags for selective purification, optimization of detergent types and concentrations, and implementation of multi-step purification protocols .

• Protein Destabilization During Storage:

  • Challenge: Loss of structural integrity over time.

  • Solution: Addition of glycerol (5-50%) to storage buffer, lyophilization with stabilizing agents like trehalose (6%), and storage at -80°C in small aliquots to avoid freeze-thaw cycles .

What quality control measures are essential for verifying recombinant atpH integrity?

Several quality control measures are essential for verifying the integrity of recombinant atpH:

• Protein Purity Assessment:

  • SDS-PAGE analysis to confirm >90% purity

  • Western blotting with specific antibodies to verify protein identity

• Structural Integrity Verification:

  • Circular dichroism spectroscopy to confirm alpha-helical secondary structure

  • Mass spectrometry to verify correct molecular weight and absence of degradation

• Functional Validation:

  • Reconstitution into liposomes to assess membrane integration

  • Proton translocation assays to verify functional capability

  • Assembly assays to confirm ability to form multimeric structures

• Stability Testing:

  • Thermal shift assays to assess protein stability

  • Time-course studies to determine shelf-life under various storage conditions

  • Freeze-thaw tolerance evaluation to establish handling protocols

• Contaminant Analysis:

  • Endotoxin testing (especially important for functional studies)

  • Host cell protein detection using sensitive analytical methods

  • Nucleic acid contamination assessment

Implementation of these quality control measures ensures that experimental results accurately reflect the properties of the target protein rather than artifacts introduced by impurities or structural alterations.

What are promising unexplored areas for atpH research in photosynthetic efficiency?

Several promising unexplored areas for atpH research could significantly advance our understanding of photosynthetic efficiency:

• C-ring Stoichiometry Engineering:

  • Investigation of whether artificially altering c-ring stoichiometry could optimize ATP synthase efficiency under specific environmental conditions

  • Development of plants with modified c-ring structures to potentially enhance photosynthetic output in agricultural applications

• Redox Regulation of ATP Synthase:

  • Exploration of how the chloroplast redox network influences ATP synthase function during light transitions

  • Investigation of potential redox-sensitive residues in atpH that might regulate activity in response to changing light conditions

• Cold Adaptation Mechanisms:

  • Detailed examination of how atpH structure and function adapt to cold environments

  • Investigation of RNA-binding proteins like CP29A and their potential regulatory effects on atpH expression and assembly

• Integration with Synthetic Biology:

  • Development of hybrid ATP synthase complexes incorporating optimized c-subunits

  • Creation of minimal synthetic ATP synthase systems to understand fundamental design principles

• Advanced Imaging Applications:

  • Application of single-molecule techniques to visualize c-ring rotation in real-time

  • Development of sensors based on c-subunit conformational changes to monitor ATP synthesis in vivo

These research directions could lead to significant advances in our understanding of photosynthetic energy conversion and potentially contribute to improved agricultural productivity through enhanced energy efficiency.

How might CRISPR/Cas9 technology advance our understanding of atpH function in vivo?

CRISPR/Cas9 technology offers powerful approaches for advancing our understanding of atpH function in vivo:

• Precise Gene Editing:

  • Introduction of specific mutations to investigate structure-function relationships

  • Creation of tagged versions of atpH for in vivo localization and interaction studies

  • Development of conditional knockouts to study essential functions

• Regulatory Element Modification:

  • Alteration of promoter regions to modulate expression levels

  • Modification of RNA processing signals to study post-transcriptional regulation

  • Engineering of reporter constructs to monitor expression dynamics

• Interspecies Comparisons:

  • Replacement of native atpH with versions from other species to study adaptation

  • Creation of chimeric proteins to identify functionally important domains

  • Introduction of variations observed in extremophile organisms to study environmental adaptations

• Multi-component Modification:

  • Simultaneous editing of multiple ATP synthase components to study cooperative functions

  • Development of optimized ATP synthase complexes for enhanced photosynthetic efficiency

  • Creation of minimal viable ATP synthase systems by systematic component modification

• In vivo Visualization Systems:

  • Integration of fluorescent reporters to monitor ATP synthase assembly and localization

  • Development of biosensors to measure ATP synthase activity in real-time

  • Creation of systems to visualize protein-protein interactions within the ATP synthase complex

Research has already demonstrated the successful application of CRISPR/Cas9 to induce mutations affecting chloroplast gene expression, revealing cold-dependent photosynthetic deficiencies . This technology promises to significantly accelerate our understanding of atpH function in the context of the complete ATP synthase complex and whole-organism physiology.