Recombinant Cuscuta obtusiflora ATP synthase subunit C, plastid (atpE)

What is the structure and function of ATP synthase subunit C in Cuscuta obtusiflora?

ATP synthase subunit C (atpE) in C. obtusiflora is a membrane-embedded protein that forms part of the F0 sector of the ATP synthase complex in chloroplasts. The protein consists of 81 amino acids with the sequence: MNPIISAASVIAAGFAVGLASIGPGIGQGTAAGRAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFI . It functions as part of an oligomeric ring that facilitates proton translocation across the thylakoid membrane, which drives the mechanical rotation coupled to ATP synthesis. This c-subunit ring is embedded in the thylakoid membrane, and its rotation is powered by the proton electrochemical gradient established during photosynthesis .

What are the alternative names and identifiers for this protein?

The ATP synthase subunit C from C. obtusiflora plastid is also known as:

• ATP synthase F0 sector subunit C

• ATPase subunit III

• Lipid-binding protein

The gene encoding this protein is named atpE, with a synonym of atpH. The UniProt accession number for this protein is A8W3H7. The full expression region spans residues 1-81 of the protein sequence .

What are the optimal methods for recombinant expression of C. obtusiflora ATP synthase subunit C?

Based on successful approaches with similar highly hydrophobic membrane proteins like ATP synthase subunit c from spinach, the following methodology is recommended:

• Gene optimization: Design a codon-optimized synthetic gene for expression in E. coli, considering the GC content and codon usage bias of the host organism.

• Fusion protein approach: Express the hydrophobic atpE as a fusion protein with a soluble partner such as maltose binding protein (MBP). This strategy has proven effective in improving solubility of membrane proteins like subunit c .

• Expression vector selection: Use a bacterial expression vector with a strong, inducible promoter (such as T7) and appropriate selection markers.

• Host strain selection: BL21 derivative E. coli strains have been successfully used for similar proteins .

• Expression conditions: Optimize temperature (typically 16-25°C for membrane proteins), inducer concentration, and duration to maximize yield while maintaining proper folding.

• Fusion protein cleavage: Include a specific protease recognition site between MBP and atpE to allow for subsequent separation .

What purification strategies yield the highest purity and functional integrity of recombinant C. obtusiflora ATP synthase subunit C?

A multi-step purification strategy is recommended:

• Initial capture: Affinity chromatography using the fusion tag (e.g., amylose resin for MBP fusion proteins)

• Fusion protein cleavage: Site-specific protease treatment to release the atpE protein from its fusion partner

• Reversed-phase chromatography: For final purification of the hydrophobic atpE protein. This approach has been successfully employed for similar ATP synthase c subunits .

• Quality assessment: Confirm the alpha-helical secondary structure using circular dichroism spectroscopy, as has been done for other c subunits .

For storage, a Tris-based buffer containing 50% glycerol is recommended, with storage at -20°C for short-term use or -80°C for extended storage. Aliquoting is advised to avoid repeated freeze-thaw cycles, with working aliquots maintained at 4°C for up to one week .

How can the functional activity of purified recombinant ATP synthase subunit C be assessed?

The functional assessment of ATP synthase subunit C can be approached through several complementary methods:

• Structural integrity verification:

  • Circular dichroism spectroscopy to confirm the expected alpha-helical secondary structure

  • Size-exclusion chromatography to assess oligomeric state and homogeneity

• Membrane incorporation assays:

  • Reconstitution into liposomes followed by proton translocation assays

  • Patch-clamp electrophysiology to measure ion channel activity

• Binding assays:

  • Interaction studies with other ATP synthase subunits using pull-down assays

  • Lipid binding assessments using fluorescence spectroscopy

• Functional complementation:

  • Genetic complementation in ATP synthase-deficient bacterial strains

These approaches provide a comprehensive assessment of both structural integrity and functional capacity of the recombinant protein.

How does the ATP synthase c-ring stoichiometry in C. obtusiflora compare to other plants, and what are the metabolic implications?

While the specific c-ring stoichiometry for C. obtusiflora ATP synthase has not been directly reported in the provided search results, this question addresses an important aspect of ATP synthase function. The number of c-subunits per ring (n) determines the H⁺/ATP ratio, which is directly related to the bioenergetic efficiency of ATP synthesis .

The c-ring stoichiometry varies across organisms:

• Bacteria: typically 10-15 subunits

• Chloroplasts: commonly 14 subunits in plants like spinach

• Mitochondria: typically 8 subunits in animals

For parasitic plants like C. obtusiflora, the c-ring stoichiometry could reflect adaptations to their unique lifestyle. A comparison with photosynthetic relatives might reveal whether the transition to parasitism has influenced this aspect of bioenergetics. The stoichiometric variation is inherently related to the metabolism of the organism, though the exact cause of this variability remains not well understood .

What evolutionary pressures have shaped the divergence of plastid genes in Cuscuta species?

Plastid genes in Cuscuta species, including those encoding ATP synthase components, have experienced unique evolutionary pressures due to the parasitic lifestyle of these plants. The research indicates:

• While some parasitic plants have lost many plastid genes, Cuscuta species retain functional copies of essential photosynthetic machinery genes, including atpE .

• Sequence divergence in plastid genes of Cuscuta appears to be driven primarily by neutral processes rather than positive selection, as evidenced by dN/dS ratios <1 in related species .

• The divergence is likely influenced by:

  • The inherently labile nature of some gene products

  • Extensive genomic rearrangement through illegitimate recombination

  • Expansions and contractions of the inverted repeat regions

• Despite sequence divergence, functional domains tend to be preserved through purifying selection, particularly for essential genes like those encoding ATP synthase components .

This evolutionary pattern suggests that while Cuscuta species have adapted to parasitism, they maintain essential plastid functions, potentially for limited photosynthesis or other metabolic processes beyond ATP production.

How do transcriptional and post-transcriptional regulatory mechanisms of atpE differ in parasitic plants like C. obtusiflora?

Research on plastid gene expression in Cuscuta and related parasitic plants reveals several insights about regulatory mechanisms:

These regulatory adaptations likely balance the reduced need for photosynthetic machinery with the maintenance of essential plastid functions in these parasitic plants.

How can structural studies of C. obtusiflora ATP synthase subunit C inform the development of selective parasitic plant control strategies?

Structural and functional studies of C. obtusiflora ATP synthase subunit C could contribute to parasitic plant control strategies through several approaches:

• Target identification: Detailed structural analysis may reveal unique features that distinguish parasitic plant ATP synthase from host plant proteins, potentially providing selective targeting opportunities.

• Structure-based inhibitor design: Atomic-level understanding of C. obtusiflora atpE structure could enable the design of molecules that specifically inhibit ATP synthase function in parasitic plants without affecting host plants.

• Metabolic vulnerability assessment: Understanding the bioenergetic parameters of C. obtusiflora ATP synthase may reveal metabolic vulnerabilities specific to parasitic plant physiology.

• Integration with existing control strategies: Knowledge about ATP synthase could complement other approaches, such as the biological control methods using Alternaria destruens for Cuscuta species .

The high host specificity demonstrated by certain biological control agents against Cuscuta suggests that targeting molecular features unique to parasitic plants can be an effective and environmentally friendly approach.

What methodological approaches can overcome the challenges in studying membrane protein complexes from parasitic plants?

Studying membrane protein complexes like ATP synthase from parasitic plants presents unique challenges that can be addressed through several methodological approaches:

• Heterologous expression systems:

  • Bacterial expression using fusion protein strategies (as demonstrated with MBP fusions)

  • Cell-free expression systems to avoid toxicity issues

  • Expression in photosynthetic hosts for functional studies

• Advanced structural biology techniques:

  • Cryo-electron microscopy for structure determination without crystallization

  • Solid-state NMR for studying membrane-embedded complexes

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

• In situ approaches:

  • Advanced microscopy techniques to study protein localization and dynamics

  • In situ hybridization methods similar to those used for studying other Cuscuta proteins

• Computational approaches:

  • Homology modeling based on structures from related species

  • Molecular dynamics simulations to study functional mechanisms

  • Evolutionary analysis to identify conserved functional domains

These approaches collectively provide a comprehensive toolkit for overcoming the challenges inherent in studying membrane proteins from organisms that are difficult to culture or obtain in large quantities.

What insights can comparative analysis of ATP synthase from photosynthetic and parasitic Cuscuta species provide about bioenergetic adaptation during the evolution of parasitism?

Comparative analysis of ATP synthase components between photosynthetic and parasitic Cuscuta species can reveal crucial insights about bioenergetic adaptation:

• Functional adaptation vs. genetic drift:

  • Comparison of selection pressures (dN/dS ratios) on atpE genes

  • Assessment of whether sequence changes alter functional parameters

  • Evaluation of conservation patterns in functional domains

• Stoichiometric variations:

  • Changes in c-ring composition affecting H⁺/ATP ratios

  • Adaptations in energy conversion efficiency

  • Balance between ATP production capacity and metabolic demand

• Regulatory adaptations:

  • Changes in gene expression patterns

  • Altered post-translational modifications

  • Modified assembly processes for the ATP synthase complex

• Metabolic integration:

  • Relationship between ATP synthesis and carbon acquisition from hosts

  • Coordination between remaining photosynthetic capacity and parasitic nutrition

  • Energy allocation patterns unique to parasitic lifestyle

This comparative approach can illuminate how essential bioenergetic machinery evolves during the transition from autotrophy to heterotrophy, potentially revealing universal principles of metabolic adaptation during major ecological transitions.

What are the common challenges in recombinant expression of ATP synthase subunit C, and how can they be addressed?

The recombinant expression of ATP synthase subunit C presents several challenges due to its hydrophobic nature and membrane localization. Common issues and their solutions include:

The fusion protein approach, particularly using MBP as demonstrated for chloroplast ATP synthase subunit c , has proven effective in overcoming many of these challenges by improving solubility and enabling efficient purification.

How can researchers optimize codon usage for maximum expression of C. obtusiflora atpE in heterologous systems?

Optimizing codon usage for C. obtusiflora atpE expression requires a systematic approach:

• Host-specific optimization:

  • Analyze codon usage bias of the expression host (e.g., E. coli BL21 derivatives)

  • Adjust rare codons in the atpE sequence to match preferred codons of the host

  • Balance GC content to improve mRNA stability and translation efficiency

• Critical region analysis:

  • Pay special attention to the first 15-25 codons, which strongly influence translation initiation

  • Avoid strong secondary structures in the mRNA, particularly near the ribosome binding site

  • Eliminate potential cryptic splice sites for eukaryotic expression systems

• Optimization parameters:

  • Codon Adaptation Index (CAI) target of >0.8 for efficient expression

  • Avoid consecutive rare codons that might cause ribosomal stalling

  • Consider harmonizing codon usage rather than maximizing it to maintain proper folding kinetics

• Experimental validation:

  • Test multiple codon-optimized variants

  • Compare expression levels and solubility

  • Assess structural integrity and function of the resulting proteins

This approach has been successfully employed for the expression of other membrane proteins, including ATP synthase components from various species .

What strategies can improve the structural and functional analysis of C. obtusiflora ATP synthase components in the context of their native membrane environment?

Analyzing ATP synthase components in their native membrane context requires specialized approaches:

• Membrane mimetic systems:

  • Reconstitution into liposomes with defined lipid composition

  • Use of nanodiscs to isolate single ATP synthase complexes

  • Incorporation into lipid bilayer nanodiscs for structural studies

• Advanced microscopy techniques:

  • Single-molecule fluorescence microscopy to observe rotational dynamics

  • Atomic force microscopy to visualize topography in membrane context

  • Super-resolution microscopy for localization studies

• Native complex isolation:

  • Develop gentle extraction protocols to maintain protein-protein interactions

  • Use detergent screens to identify optimal solubilization conditions

  • Employ gradient centrifugation or native electrophoresis for complex separation

• In situ structural biology:

  • Electron tomography of membrane sections

  • In-cell NMR for structural information in cellular context

  • Cross-linking mass spectrometry to map protein interactions

• Functional assessment:

  • Develop electrochemical assays to measure proton translocation

  • Use ATP synthesis assays in reconstituted systems

  • Apply patch-clamp techniques to measure channel properties