Recombinant Nasturtium officinale ATP synthase subunit c, chloroplastic (atpH)

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

ATP synthase subunit c (atpH) is a critical component of the F₀ sector of ATP synthase in chloroplasts. This hydrophobic protein forms the c-ring structure within the membrane-embedded portion of the enzyme. The c-ring functions as a proton channel and rotary motor that converts the energy of the proton gradient across the thylakoid membrane into mechanical energy. This rotation is mechanically coupled to conformational changes in the F₁ catalytic domain, which drives ATP synthesis from ADP and inorganic phosphate .

The structure of subunit c consists primarily of two transmembrane α-helices connected by a polar loop region. Multiple copies of subunit c (typically 8-15 depending on the species) assemble into a ring structure. The exact number of c subunits in Nasturtium officinale ATP synthase has not been definitively established in the available research, but structural studies would be necessary to determine this species-specific characteristic.

How does Nasturtium officinale ATP synthase subunit c differ from other plant species?

Sequence analysis of the atpH gene from Nasturtium officinale reveals several conserved residues that are crucial for function, particularly the proton-binding glutamate or aspartate residue (similar to Glu 59 in bovine or Asp 61 in E. coli) that is essential for proton translocation through the c-ring . While the general structure is conserved across species, subtle variations in the amino acid sequence may affect proton binding affinity, c-ring stability, and interactions with other ATP synthase subunits.

Comparative genomic analysis would be required to fully characterize the unique features of N. officinale ATP synthase subunit c. Such analysis should focus on residues involved in proton binding, subunit-subunit interactions, and potential post-translational modifications that might be specific to this species.

What expression systems are most suitable for recombinant production of chloroplastic ATP synthase subunit c?

The recombinant production of membrane proteins like ATP synthase subunit c presents significant challenges due to their hydrophobic nature. Based on current methodologies for similar proteins, several expression systems can be considered:

• Escherichia coli expression systems: These systems offer high yield and simplicity but may require optimization for membrane protein expression. For ATP synthase subunit c, E. coli expression systems have been successfully employed with modifications such as fusion partners to increase solubility .

• Yeast expression systems: Saccharomyces cerevisiae or Pichia pastoris can provide eukaryotic post-translational modifications and membrane insertion machinery, potentially improving the folding of plant chloroplastic proteins.

• Plant-based expression systems: These may provide the most native-like environment for chloroplastic proteins but typically yield lower protein amounts.

The choice of expression system should consider factors such as required yield, post-translational modifications, and downstream applications. For structural studies requiring high protein quantities, E. coli systems with optimization for membrane protein expression would be most practical.

What are the optimal conditions for maximizing expression yield of recombinant Nasturtium officinale ATP synthase subunit c?

Optimizing expression conditions for recombinant ATP synthase subunit c requires a systematic approach, ideally implementing Design of Experiment (DoE) methodologies rather than traditional one-factor-at-a-time approaches . Key parameters to optimize include:

• Temperature: Lower temperatures (16-25°C) often improve membrane protein folding and reduce inclusion body formation.

• Induction conditions: For inducible systems, optimizing inducer concentration and induction timing is critical. Low inducer concentrations and induction during late log phase may improve proper membrane insertion.

• Media composition: Supplementing growth media with glycerol (0.5-2%) can improve membrane protein expression.

• Co-expression with chaperones: Molecular chaperones like GroEL/GroES may assist in proper folding.

A central composite circumscribed (CCC) design experiment, similar to that used for other recombinant proteins, would allow simultaneous optimization of multiple parameters . This approach would identify not only the main effects but also interaction effects between variables that influence expression yield.

What purification protocol yields the highest purity for recombinant ATP synthase subunit c while maintaining its native structure?

Purification of recombinant ATP synthase subunit c presents unique challenges due to its hydrophobic nature and tendency to form aggregates. A multi-step purification protocol, optimized through DoE approaches, would yield the best results:

Step 1: Membrane Extraction and Solubilization

• Isolate membrane fractions from expression host

• Solubilize membranes using detergents (commonly n-dodecyl-β-D-maltoside or digitonin for ATP synthase components)

Step 2: Affinity Chromatography

• If expressed with an affinity tag, use appropriate affinity chromatography (His-tag or other)

• Optimize elution conditions using DoE approach to minimize aggregation

• For example, adjusting pH to 2.9-3.0 with specific additives like trehalose (32-35% w/v) has been shown to reduce aggregation in other recombinant proteins

Step 3: Ion Exchange Chromatography

• Apply to cation exchange matrix (e.g., Capto S) at optimized pH and conductivity

• The optimal conditions would likely be in pH range of 5.9-6.1 and loading conductivity of 5-12.5 mS/cm based on similar proteins

Step 4: Size Exclusion Chromatography

• Final polishing step to separate monomeric protein from aggregates

• Buffer conditions should be optimized to maintain protein stability

This protocol should yield protein with >95% purity while maintaining the native structure. The use of appropriate detergents throughout the purification process is critical for membrane protein stability.

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

Assessing the functional integrity of purified ATP synthase subunit c requires multiple complementary approaches:

• Structural Integrity Assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure content

  • Thermal shift assays to evaluate protein stability

  • Limited proteolysis to assess proper folding

• Oligomeric State Analysis:

  • Analytical size exclusion chromatography to determine if the protein forms the expected oligomeric structures

  • Native PAGE to analyze complex formation

  • Analytical ultracentrifugation to determine the precise oligomeric state

• Functional Assays:

  • Reconstitution into liposomes and measurement of proton translocation

  • Co-reconstitution with other ATP synthase subunits to assess complex formation

  • Measurement of ATP synthesis/hydrolysis when integrated with other subunits

• Binding Studies:

  • Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to measure interactions with other ATP synthase subunits

  • Studies with specific inhibitors like oligomycin or dicyclohexylcarbodiimide (DCCD) that target the Fo portion

A comprehensive assessment would include multiple methods from the above categories to ensure both structural and functional integrity of the purified protein.

How can recombinant Nasturtium officinale ATP synthase subunit c be used to study the plant's unique antioxidant properties?

Nasturtium officinale (watercress) demonstrates significant antioxidant properties, as evidenced by its ability to reduce oxidative stress in hypercholesterolemic conditions . To investigate the potential relationship between ATP synthase function and these antioxidant properties, researchers could:

• Investigate energy metabolism coupling: Examine how ATP production via ATP synthase affects cellular redox state in N. officinale. Recombinant subunit c could be used in reconstitution experiments to measure ATP synthesis efficiency under various oxidative conditions.

• Study post-translational modifications: Analyze whether the subunit c undergoes specific post-translational modifications (such as oxidation or glutathionylation) under oxidative stress that might regulate ATP synthase activity.

• Cross-species comparative studies: Compare the sequence and functional properties of ATP synthase subunit c from N. officinale with those from plants lacking similar antioxidant properties to identify unique structural or functional adaptations.

• Regulatory interactions: Identify potential protein-protein interactions between ATP synthase components and antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), or glutathione peroxidase (GPx), which show altered activities in N. officinale under stress conditions .

The following table summarizes potential experimental approaches:

What are the appropriate controls and validation methods when studying recombinant ATP synthase subunit c function?

Rigorous experimental design for studying recombinant ATP synthase subunit c function requires appropriate controls and validation methods:

• Negative Controls:

  • Expression and purification of a non-functional mutant (e.g., mutation of the critical proton-binding residue)

  • Purification from expression system without the subunit c gene

  • Reconstitution experiments with heat-denatured protein

• Positive Controls:

  • Parallel expression and characterization of well-studied ATP synthase subunit c from model organisms (E. coli, spinach)

  • Complementation studies in appropriate knockout systems

• Validation of Structure and Assembly:

  • Cross-linking studies to confirm proper subunit interactions

  • Electron microscopy to visualize assembled complexes

  • Mass spectrometry to confirm protein identity and modifications

• Functional Validation:

  • Inhibitor studies using specific ATP synthase inhibitors

  • Measurement of proton translocation using pH-sensitive fluorescent dyes

  • Comparison of ATP synthesis/hydrolysis rates with native ATP synthase

When designing experiments, researchers should implement a Design of Experiment approach rather than one-factor-at-a-time methods to comprehensively understand parameter interactions and optimize experimental conditions .

How can researchers overcome aggregation issues during recombinant ATP synthase subunit c purification?

Aggregation is a common challenge when working with hydrophobic membrane proteins like ATP synthase subunit c. Several strategies can minimize aggregation:

• Optimization of Detergent Types and Concentrations:

  • Systematic screening of detergents (mild non-ionic detergents like DDM, LMNG, or digitonin)

  • Maintaining detergent concentration above critical micelle concentration (CMC) throughout purification

  • Consideration of detergent mixtures or novel amphipathic polymers (amphipols)

• Buffer Optimization Using DoE Approach:

  • Identification of critical factors affecting aggregation

  • For example, in similar recombinant protein purifications, pH (2.9-3.0) and trehalose concentration (32-35% w/v) were identified as critical parameters for reducing aggregation

  • Implementation of CCC design to identify optimal conditions

• Addition of Stabilizing Agents:

  • Glycerol (10-20%) to stabilize hydrophobic regions

  • Specific lipids that may be required for proper folding

  • Cholesterol hemisuccinate or other membrane mimetics

• Temperature Management:

  • Maintaining low temperatures (4°C) during purification

  • Avoiding freeze-thaw cycles by aliquoting purified protein

• Prevention of Oxidation:

  • Addition of reducing agents like DTT or TCEP

  • Working under nitrogen atmosphere for sensitive preparations

The effectiveness of these approaches should be monitored using dynamic light scattering (DLS) and analytical size exclusion chromatography to quantify the monodispersity of the protein preparation.

What are the most common mistakes in experimental design when working with recombinant chloroplastic proteins, and how can they be avoided?

Researchers frequently encounter several pitfalls when working with recombinant chloroplastic proteins like ATP synthase subunit c:

• Inappropriate Expression System Selection:

  • Mistake: Choosing expression systems without considering the unique requirements of chloroplastic proteins

  • Solution: Evaluate multiple expression systems with pilot experiments; consider chloroplast transit peptide effects on expression

• Inadequate Detergent Selection:

  • Mistake: Using harsh detergents that may denature the protein or insufficient detergent concentrations

  • Solution: Systematic detergent screening; monitoring protein stability in each detergent; considering native lipid environment

• Neglecting Redox Conditions:

  • Mistake: Failing to control oxidation state during purification

  • Solution: Maintain reducing conditions; consider the native redox environment of chloroplasts

• One-dimensional Optimization Approach:

  • Mistake: Using one-factor-at-a-time optimization instead of statistical experimental design

  • Solution: Implement DoE approaches like fractional factorial or CCC designs to identify optimal conditions and interaction effects

• Insufficient Functional Validation:

  • Mistake: Relying solely on protein yield and purity without confirming functional integrity

  • Solution: Implement multiple complementary functional assays to confirm protein activity in a native-like environment

The table below summarizes common issues and their solutions:

How does research on recombinant ATP synthase subunit c contribute to understanding plant bioenergetics in stress conditions?

Research on recombinant ATP synthase subunit c from Nasturtium officinale can significantly enhance our understanding of plant bioenergetics, particularly under stress conditions:

• Energy Production Under Oxidative Stress:

  • ATP synthase function may be modified under oxidative stress conditions, affecting energy balance

  • N. officinale shows enhanced antioxidant enzyme activities (CAT, SOD) and modulated glutathione-related enzyme activities (GPx, GR) under stress conditions

  • Recombinant subunit c can be used to study how ATP production is maintained during these stress responses

• Regulatory Mechanisms:

  • ATP synthase regulation may be a key component of plant adaptation to environmental stressors

  • Studies with recombinant subunit c can reveal how specific post-translational modifications affect enzyme function

  • The reversibility of ATP synthase (acting as ATPase under certain conditions) may be important for cellular energy homeostasis

• Evolutionary Adaptations:

  • Comparative studies of ATP synthase components from different plant species can reveal evolutionary adaptations

  • N. officinale's robust antioxidant system may be linked to specific adaptations in energy metabolism

• Metabolic Integration:

  • ATP synthase activity is integrated with other metabolic pathways

  • Recombinant protein studies can help map the interplay between energy production and consumption pathways during stress

Understanding these aspects could lead to applications in improving crop stress tolerance or harnessing natural antioxidant systems for biotechnological applications.

What methodological approaches from recombinant ATP synthase research can be applied to other challenging membrane proteins?

The methodologies developed for recombinant ATP synthase subunit c research have broader applications for other challenging membrane proteins:

• Statistical Experimental Design Approaches:

  • The DoE techniques used for optimizing purification conditions can be applied to other membrane proteins

  • Fractional factorial designs for initial screening followed by CCC designs for fine-tuning represent a powerful approach that reduces experimental burden while identifying optimal conditions

• Detergent Screening and Stabilization Strategies:

  • Systematic approaches to detergent selection and optimization

  • Use of stabilizing agents like trehalose that proved effective for recombinant proteins

  • Application of pH optimization techniques that maintain protein stability while reducing aggregation

• Functional Reconstitution Methods:

  • Techniques for reconstituting membrane proteins into liposomes or nanodiscs

  • Methods for assessing functional integrity in membrane environments

  • Approaches for co-reconstitution with interacting partners

• Multi-step Purification Optimization:

  • Integration of multiple chromatographic steps with condition optimization at each stage

  • Buffer exchange strategies that maintain protein stability

  • Scale-up methods that preserve protein quality at larger scales

The lessons learned from working with challenging proteins like ATP synthase subunit c provide valuable insights for the broader field of membrane protein biochemistry, potentially improving success rates for structural and functional studies of diverse membrane proteins.

What are the emerging technologies that could advance research on recombinant ATP synthase components?

Several cutting-edge technologies show promise for advancing research on recombinant ATP synthase components:

• Cryo-Electron Microscopy (Cryo-EM):

  • Allows visualization of membrane proteins in near-native states without crystallization

  • Can resolve different conformational states of ATP synthase during catalytic cycle

  • Recent advances in detector technology and image processing have dramatically improved resolution

• Native Mass Spectrometry:

  • Enables analysis of intact membrane protein complexes

  • Can provide insights into subunit stoichiometry and ligand binding

  • Recent developments allow membrane proteins to be analyzed in detergent micelles or nanodiscs

• Single-Molecule Techniques:

  • Methods like fluorescence resonance energy transfer (FRET) can track conformational changes

  • Optical tweezers can directly measure the mechanical force generated by ATP synthase rotation

  • These approaches provide insights into the dynamics not accessible through ensemble measurements

• Cell-Free Expression Systems:

  • Allow rapid production of membrane proteins

  • Can incorporate non-natural amino acids for site-specific labeling

  • Enable direct integration into membrane mimetics during synthesis

• Computational Approaches:

  • Molecular dynamics simulations of the c-ring in membrane environments

  • Machine learning for prediction of optimal expression and purification conditions

  • Systems biology modeling of ATP synthase in the context of cellular energetics

These technologies, applied to recombinant ATP synthase subunit c from Nasturtium officinale, could reveal unprecedented details about structure-function relationships and species-specific adaptations.

How might research on Nasturtium officinale ATP synthase components inform biotechnological applications?

Research on Nasturtium officinale ATP synthase components has several potential biotechnological applications:

• Bioenergetic Applications:

  • Design of biomimetic energy conversion systems based on ATP synthase principles

  • Development of nanoscale rotary motors inspired by the c-ring structure

  • Creation of biosensors for proton gradients or ATP levels

• Therapeutic Applications:

  • N. officinale's demonstrated antioxidant and lipid-lowering properties could be linked to energy metabolism

  • Understanding the molecular basis could lead to novel therapeutic strategies

  • Potential development of compounds that modulate ATP synthase activity for medical applications

• Agricultural Applications:

  • Engineering crops with enhanced stress tolerance based on insights from N. officinale energy metabolism

  • Improving photosynthetic efficiency through modifications to ATP synthase components

  • Developing crops with enhanced nutritional profiles based on N. officinale's beneficial properties

• Protein Engineering Applications:

  • Using insights from recombinant protein production to develop improved expression systems

  • Creating chimeric proteins with enhanced stability or activity

  • Designing modified ATP synthase components for specific biotechnological applications

The table below summarizes potential applications and their foundational research requirements:

These applications represent the translation potential of fundamental research on ATP synthase components from N. officinale, highlighting the value of this research beyond basic science understanding.