Recombinant Gnetum parvifolium ATP synthase subunit c, chloroplastic (atpH)

How does the atpH gene in Gnetum parvifolium compare to other plant species?

The atpH gene in Gnetum parvifolium encodes the ATP synthase subunit c protein (Uniprot ID: A6BM12), also known as ATP synthase F(0) sector subunit c, ATPase subunit III, or F-type ATPase subunit c . While the basic structure and function of ATP synthase are conserved across species, there are notable variations in subunit composition and stoichiometry.

Compared to other ATP synthases, G. parvifolium's enzyme is particularly interesting due to Gnetum's unique phylogenetic position. As a member of Gnetophyta, G. parvifolium represents a small, distinct group with a controversial evolutionary placement in the plant kingdom . This makes its ATP synthase components valuable for comparative studies across plant lineages.

What are the optimal methods for expressing and purifying recombinant G. parvifolium ATP synthase subunit c?

For efficient expression and purification of recombinant G. parvifolium ATP synthase subunit c, the following methodology has proven effective:

Expression System:

• Use E. coli as the expression host for the recombinant protein

• Express the full-length protein (amino acids 1-81) with an N-terminal His-tag for easier purification

Purification Protocol:

• Perform affinity chromatography using the N-terminal His-tag

• Store the purified protein in a Tris-based buffer with 50% glycerol to maintain stability

• For long-term storage, maintain at -20°C or -80°C

• Avoid repeated freeze-thaw cycles; instead, prepare working aliquots and store at 4°C for up to one week

When expressing membrane proteins like ATP synthase subunit c, careful optimization of expression conditions (temperature, induction time, inducer concentration) is crucial to prevent formation of inclusion bodies and ensure proper folding.

How can researchers effectively analyze the structure and function of recombinant G. parvifolium ATP synthase subunit c?

Several complementary techniques can be employed to analyze the structure and function of recombinant G. parvifolium ATP synthase subunit c:

Structural Analysis:

• Cryo-electron microscopy (Cryo-EM): This technique has been successfully used to resolve high-resolution structures of ATP synthase complexes, including rotational states in the F₁ sector and the arrangement of c-subunits in the c-ring

• FTIR Spectroscopy: Can be used to analyze secondary structure elements and detect functional groups within the protein. Peaks in the range of 1000-1700 cm⁻¹ can provide information about protein structural elements

Functional Analysis:

• ATP Synthesis/Hydrolysis Assays: Measure the rate of ATP synthesis or hydrolysis using reconstituted proteoliposomes

• Proton Translocation Assays: Use pH-sensitive fluorescent dyes to monitor proton movement across membranes

• Rotation Assays: Fluorescently label components to visualize rotation, similar to studies in bacterial F₁ that showed rotation speeds exceeding 130Hz (approximately 400 ATP molecules synthesized per second)

What experimental designs are most appropriate for studying ATP synthase function in plant systems?

When investigating ATP synthase function in G. parvifolium and other plant systems, researchers should consider these experimental approaches:

Fully Experimental Designs:

• Randomized Controlled Trials (RCTs): Useful for comparing different conditions or treatments affecting ATP synthase function, such as testing the effects of environmental stressors on enzyme activity

Quasi-Experimental Designs:

• Pre-post designs with non-equivalent control groups: Suitable for studying ATP synthase before and after exposure to different conditions

• Interrupted time series (ITS): Valuable for monitoring ATP synthase activity over time with periodic interventions

• Stepped wedge designs: Appropriate when interventions need to be introduced in a staggered fashion across different experimental groups

Single Subject Experimental Designs (SSEDs):

• These can be used when studying specific modifications to ATP synthase components, though they require reversibility of the intervention

Selection of the appropriate design depends on the specific research question, available resources, and ethical considerations when using plant material from rare species like G. parvifolium.

How does the c-ring structure in G. parvifolium ATP synthase contribute to its functional properties?

The c-ring in ATP synthase is composed of multiple c-subunits arranged in a circular formation within the membrane. This structure is critical for energy conversion and has several important functional implications:

Structure-Function Relationship:

• The c-ring acts as a rotor that converts the energy from proton flow across the membrane into mechanical rotation

• This rotation is transmitted via the central stalk (γ-subunit) to the F₁ catalytic head, driving conformational changes that synthesize ATP

Stoichiometry and Efficiency:

• The number of c-subunits in the rotor ring is an adaptation to the physiological environment

• This stoichiometry determines the ion-to-ATP ratio, affecting the enzyme's efficiency as either an ATP synthase or an ion pump

• For example, a larger c-ring with more subunits would have a higher ion-to-ATP ratio, making the enzyme more efficient as an ion pump

While specific data on G. parvifolium's c-ring stoichiometry is not available, variations in this parameter could reflect adaptations to the unique ecological niches of this plant species.

What are the implications of studying ATP synthase from evolutionarily unique plants like G. parvifolium?

G. parvifolium belongs to Gnetophyta, a small group with a controversial phylogenetic position, making its ATP synthase particularly valuable for evolutionary studies . Research on this protein has several significant implications:

Evolutionary Insights:

• Comparing ATP synthase components across plant lineages can help resolve phylogenetic relationships

• Structural variations may provide evidence for adaptive evolution in response to different environmental pressures

• The c-ring stoichiometry, which typically doesn't vary within a species but differs between species, can offer insights into evolutionary adaptations

Structural Biology Advances:

• Novel structural features in G. parvifolium ATP synthase could reveal alternative mechanisms for energy conversion

• Comparative analysis with other plant ATP synthases could identify conserved functional domains versus lineage-specific innovations

• Understanding how membrane lipids interact with ATP synthase in different plant lineages can illuminate co-evolution of proteins with their membrane environment

This research contributes to our fundamental understanding of energy metabolism evolution across the plant kingdom and may reveal unique adaptations in the Gnetophyta lineage.

How might environmental factors affect ATP synthase expression and function in G. parvifolium?

Environmental factors likely influence ATP synthase expression and function in G. parvifolium, similar to their effects on other metabolic pathways in this plant:

Temperature Effects:

• High temperature has been shown to induce expression of genes involved in secondary metabolite biosynthesis in G. parvifolium

• Similarly, temperature stress might alter ATP synthase expression to meet changing energy demands

UV Radiation Response:

• UV-C exposure strongly induces gene expression changes in G. parvifolium

• This environmental stress might also affect ATP synthase function or abundance to support stress response mechanisms

Experimental Approach to Study Environmental Effects:

Understanding how environmental factors influence ATP synthase could provide insights into G. parvifolium's adaptation mechanisms and energy management strategies under stress conditions.

How can recombinant G. parvifolium ATP synthase components be used in biotechnological applications?

Recombinant ATP synthase components from G. parvifolium have several potential biotechnological applications:

Structural Biology Tools:

• The recombinant protein can serve as a model system for studying membrane protein structure and function

• His-tagged proteins facilitate purification and crystallization for structural studies

Bioenergetic Applications:

• ATP synthase components could potentially be incorporated into artificial membrane systems for energy conversion applications

• Understanding the unique properties of G. parvifolium ATP synthase might inspire biomimetic energy conversion technologies

Comparative Biochemistry:

• The recombinant protein enables direct comparison with ATP synthase components from other species

• This comparative approach can identify unique features that might have biotechnological value

Future research should explore the potential for engineering ATP synthase components with enhanced properties for specific biotechnological applications.

What are the methodological challenges in studying membrane protein complexes like ATP synthase from rare plant species?

Researchers face several methodological challenges when studying ATP synthase from rare plant species like G. parvifolium:

Technical Challenges:

• Limited Source Material: Rare plant species provide limited biomass for protein extraction

• Membrane Protein Solubility: Hydrophobic membrane proteins like ATP synthase subunit c are challenging to solubilize while maintaining native structure

• Complex Assembly: The multi-subunit nature of ATP synthase makes reconstitution of functional complexes difficult

• Lipid Requirements: ATP synthase function depends on specific lipid environments that may be difficult to replicate in vitro

Methodological Solutions:

• Recombinant Expression: Express individual subunits or subcomplexes in bacterial systems with appropriate tags

• Detergent Optimization: Screen multiple detergents for optimal solubilization (e.g., n-dodecyl β-D-maltoside and glyco-diosgenin have been successful for ATP synthase purification)

• Lipid Reconstitution: Incorporate purified components into liposomes with defined lipid composition

• Advanced Imaging: Use cryo-EM and other advanced structural techniques that require relatively small amounts of material

Addressing these challenges requires multidisciplinary approaches combining molecular biology, biochemistry, and structural biology techniques.

How can the study of G. parvifolium ATP synthase contribute to our understanding of plant adaptation and evolution?

Research on G. parvifolium ATP synthase can provide valuable insights into plant adaptation and evolution in several ways:

Evolutionary Context:

• Gnetophyta occupies a unique phylogenetic position, making comparative studies of its energy metabolism particularly informative for understanding plant evolution

• Analysis of ATP synthase structure and function across diverse plant lineages can help resolve evolutionary relationships and identify convergent adaptations

Adaptation Mechanisms:

• Variations in ATP synthase components may reflect adaptations to different environmental niches

• The c-ring stoichiometry, which affects the bioenergetic efficiency of ATP synthase, can be an important adaptation to different energy demands

Research Approach for Evolutionary Studies:

• Compare ATP synthase gene sequences across plant lineages

• Analyze selection pressures on ATP synthase genes in different plant groups

• Reconstruct ancestral sequences to trace the evolution of key functional domains

• Correlate structural variations with ecological adaptations

This research not only advances our understanding of plant evolution but may also reveal adaptive mechanisms that could be valuable for crop improvement in the face of environmental challenges.