Recombinant Oedogonium cardiacum ATP synthase subunit c, chloroplastic (atpH)

What is the structural characterization of Recombinant Oedogonium cardiacum ATP synthase subunit c, chloroplastic (atpH)?

Recombinant Oedogonium cardiacum ATP synthase subunit c is a transmembrane protein encoded by the atpH gene in the chloroplast genome. The protein consists of 83 amino acids with the sequence: MNPLIAASSVIAAGLAIGLAAIGPGVGQGTVAGNAVEGIARQPEAEGKIRGTLLLSFAFMESLTIYGLVVALALLFANPFVGA . As a component of the F0 sector of ATP synthase, this protein primarily functions within the membrane-embedded portion of the complex. The recombinant form is typically produced with an N-terminal 10xHis-tag to facilitate purification, though the tag configuration may vary depending on protein-tag stability considerations . The protein's hydrophobic nature is evident from its amino acid composition, featuring multiple alanine and leucine residues, which are characteristic of transmembrane domains in ATP synthase subunit c proteins.

How does Oedogonium cardiacum ATP synthase subunit c contribute to chloroplast genome architecture?

The atpH gene encoding ATP synthase subunit c is part of the highly compact Oedogonium cardiacum chloroplast genome, which has a circular structure of 196,547 bp with an A+T content of 70.5% . This genome displays an atypical quadripartite structure with two identical copies of a large 35,492-bp inverted repeat (IR) separated by single-copy regions of 80,363 and 45,200 bp . Unlike typical chloroplast genomes, the gene partitioning pattern in Oedogonium deviates considerably from ancestral patterns. The genome's architecture reflects significant evolutionary events, including gene rearrangements and horizontal gene transfers. ATP synthase subunit c is among the 99 conserved genes found in this compact genome, where intergenic spacers account for only 22.6% of the total sequence - the lowest proportion observed among photosynthetic chlorophytes of the UTC (Ulvophyceae, Trebouxiophyceae or Chlorophyceae) clade .

What are the optimal storage and handling conditions for recombinant atpH protein?

For recombinant Oedogonium cardiacum ATP synthase subunit c, proper storage is critical to maintain structural integrity and functionality. The recommended storage temperature is -20°C for routine storage, while extended preservation should be at -20°C or -80°C . Working aliquots can be maintained at 4°C for up to one week to minimize freeze-thaw cycles . Researchers should avoid repeated freezing and thawing as this can significantly compromise protein stability and function . Given its transmembrane nature, the protein is typically stored in a Tris-based buffer with 50% glycerol that has been optimized for stability . When planning experiments, researchers should consider preparing multiple small aliquots during initial reconstitution to minimize the need for repeated freezing. The shelf life varies depending on storage conditions: liquid form preparations typically remain stable for approximately 6 months at -20°C/-80°C, while lyophilized preparations can maintain integrity for up to 12 months under the same conditions .

What evolutionary insights can be derived from studying atpH in Oedogonium cardiacum?

The atpH gene in Oedogonium cardiacum provides significant evolutionary insights when compared with other chlorophycean species. The Oedogonium chloroplast genome shares unique genetic characteristics with Stigeoclonium, including the retention of psaM, rpl32, and trnL(caa), as well as the loss of petA . These shared features bolster evidence for a close phylogenetic relationship between the Oedogoniales and Chaetophorales orders. Additionally, the Oedogonium genome displays evidence of horizontal gene transfer events, with three genes acquired in a 10-kb region of its exceptionally large inverted repeat . This includes the identification of int and dpoB genes, which show high similarity to genes not typically found in chloroplast DNA. These findings provide clear evidence that novel genes were acquired through horizontal transfers, possibly from a mitochondrial genome donor. These evolutionary patterns indicate that the ATP synthase complex in Oedogonium has evolved within the context of significant genomic restructuring events that characterize the divergence of major chlorophycean lineages.

How does recombinant protein expression affect the structure-function relationship of atpH?

The recombinant expression of Oedogonium cardiacum ATP synthase subunit c introduces important considerations for structure-function analyses. The addition of tags (typically an N-terminal 10xHis-tag) facilitates purification but may influence protein folding or oligomerization . Given that native ATP synthase subunit c forms a ring structure in the F0 sector, researchers must carefully evaluate whether the recombinant form maintains proper assembly capabilities. The E. coli expression system commonly used for producing this protein may not replicate all post-translational modifications present in the native chloroplast environment . Researchers should implement validation protocols to confirm that the recombinant protein retains essential structural characteristics. This validation typically involves circular dichroism spectroscopy to assess secondary structure content, particularly the alpha-helical domains critical for membrane insertion. Additionally, functional assays measuring proton translocation or ATP synthase activity using reconstituted proteoliposomes can verify whether the recombinant protein maintains native functional properties despite the expression system differences.

What methodologies are most effective for functional characterization of recombinant Oedogonium cardiacum ATP synthase subunit c?

Functional characterization of recombinant Oedogonium cardiacum ATP synthase subunit c requires specialized approaches due to its transmembrane nature. The most effective methodology involves a multi-step process beginning with protein reconstitution into liposomes or nanodiscs to create a membrane-like environment. This is typically achieved using a detergent-mediated reconstitution protocol with phospholipids that mimic the chloroplast membrane composition. Following reconstitution, researchers can employ several complementary techniques:

• Proton conductance assays using pH-sensitive fluorescent dyes (such as ACMA or pyranine) to monitor proton movement through the c-ring

• ATP synthesis/hydrolysis coupling measurements in reconstituted systems containing both F0 and F1 sectors

• Single-molecule FRET to analyze conformational changes during proton translocation

• Cryo-electron microscopy to visualize the assembled c-ring structure with high resolution

For interaction studies with other ATP synthase subunits, researchers should consider surface plasmon resonance (SPR) or microscale thermophoresis (MST) techniques, which can measure binding affinities while requiring minimal protein amounts. Site-directed mutagenesis of key residues involved in proton binding (particularly glutamate or aspartate residues in the transmembrane region) can provide insight into specific functional mechanisms when combined with the above approaches.

How can researchers address challenges in studying membrane protein-lipid interactions of atpH?

Studying membrane protein-lipid interactions for ATP synthase subunit c presents several methodological challenges that researchers must address through specialized approaches. The hydrophobic nature of this protein can lead to aggregation and misfolding during purification and reconstitution. To overcome these obstacles, researchers should implement the following strategies:

• Use mild detergents (such as DDM or LMNG) during initial extraction and purification steps, then carefully exchange to lipid environments

• Employ native nanodiscs or SMALP (styrene maleic acid lipid particles) technologies that preserve native lipid interactions during protein extraction

• Utilize solid-state NMR spectroscopy to analyze protein-lipid interactions in membrane environments

• Apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify lipid-binding regions

For mapping specific lipid interaction sites, researchers can employ photoactivatable and clickable lipid analogs combined with mass spectrometry. Molecular dynamics simulations provide complementary insights by predicting lipid binding sites and analyzing how different lipid compositions affect protein structure and dynamics. Additionally, researchers should consider the unique lipid composition of chloroplast membranes when designing reconstitution experiments, particularly incorporating glycolipids and PG (phosphatidylglycerol) that are abundant in thylakoid membranes and may be essential for proper function of this ATP synthase component.

What implications do horizontal gene transfer events in the Oedogonium cardiacum chloroplast genome have for ATP synthase evolution?

The discovery of horizontal gene transfer (HGT) events in the Oedogonium cardiacum chloroplast genome has profound implications for our understanding of ATP synthase evolution. While the atpH gene itself does not appear to be acquired through HGT, the identification of int and dpoB genes acquired through horizontal transfer in a 10-kb region of the inverted repeat suggests significant genomic plasticity in this organism . This genomic flexibility may have influenced the evolution of the entire ATP synthase complex. The evolutionary trajectory of ATP synthase components might be indirectly affected by these HGT events through several mechanisms:

• Altered regulatory networks controlling ATP synthase gene expression

• Modified genetic contexts that influence gene expression patterns

• Introduction of selective pressures on other components to accommodate functional interactions

These findings challenge the traditional view of chloroplast genome evolution as primarily driven by vertical inheritance and suggest a more complex evolutionary history involving genetic exchange across diverse lineages. Researchers investigating ATP synthase evolution should consider analyzing selection pressures on atpH and other ATP synthase components in Oedogonium compared to related species that lack these HGT events. The close relationship between Oedogonium and Stigeoclonium at the gene content and gene order levels provides a valuable comparative framework for such analyses . Additionally, researchers should investigate whether these HGT events coincide temporally with changes in ATP synthase structure or function, potentially using phylogenetic dating methods combined with structural predictions.

How can structural studies of Oedogonium cardiacum ATP synthase subunit c inform bioenergetic models in green algae?

• High-resolution structural determination through X-ray crystallography or cryo-electron microscopy

• Analysis of the number of c-subunits per ring, which influences the thermodynamic efficiency of ATP synthesis

• Identification of key residues involved in proton binding and translocation

• Computational modeling of proton movement through the assembled c-ring

Comparative structural analyses between Oedogonium and other chlorophycean algae can reveal adaptations related to specific ecological niches or photosynthetic efficiencies. Researchers should focus on potential structural adaptations that may optimize ATP synthase performance under various light conditions or during state transitions in photosynthesis. By integrating structural data with functional measurements of proton translocation and ATP synthesis rates, researchers can develop comprehensive bioenergetic models that account for species-specific variations in energy coupling efficiencies. These models would be particularly valuable for understanding how different green algal lineages have optimized their energy conversion processes through evolutionary time.

What experimental approaches can elucidate the assembly process of ATP synthase incorporating the recombinant atpH protein?

Elucidating the assembly process of ATP synthase incorporating recombinant Oedogonium cardiacum ATP synthase subunit c requires sophisticated experimental approaches that track protein-protein interactions and complex formation. Researchers should implement the following methodological strategies:

• In vitro reconstitution systems using purified components combined with time-resolved structural analysis

• Pulse-chase experiments with isotopically labeled proteins to track assembly kinetics

• Chemical crosslinking combined with mass spectrometry (XL-MS) to identify interaction interfaces

• Single-molecule fluorescence microscopy to visualize assembly intermediates

The c-subunit ring typically forms early in ATP synthase assembly, serving as a foundation for subsequent incorporation of other F0 components. To study this process, researchers can employ fluorescently labeled variants of the recombinant protein and monitor oligomerization using fluorescence correlation spectroscopy (FCS) or single-molecule FRET. Additionally, researchers should develop strategies to differentiate between homo-oligomeric interactions (c-c subunit interactions) and hetero-oligomeric interactions (c-subunit with other ATP synthase components). Native mass spectrometry is particularly valuable for analyzing intact complexes and subcomplexes during assembly. Comparing assembly pathways between recombinant systems and native chloroplast environments may reveal important chaperone or assembly factor requirements that could be targeted in future studies aimed at improving recombinant expression and functional reconstitution.

What are the critical parameters for optimizing expression of recombinant Oedogonium cardiacum ATP synthase subunit c?

Optimizing expression of recombinant Oedogonium cardiacum ATP synthase subunit c requires careful consideration of expression systems, culture conditions, and purification strategies. The E. coli expression system is commonly used , but researchers must address several key parameters:

Additionally, researchers should consider codon optimization of the gene sequence for E. coli expression and explore fusion partners that can enhance solubility while maintaining function. For challenging expressions, alternative systems such as cell-free expression in the presence of lipid nanodiscs can be employed to directly incorporate the protein into membrane-like environments during synthesis, potentially improving folding and functionality of this hydrophobic protein.

How should researchers design functional assays to evaluate ATP synthase activity incorporating recombinant atpH?

Designing functional assays for ATP synthase incorporating recombinant Oedogonium cardiacum ATP synthase subunit c requires consideration of both isolated subunit properties and integrated complex function. Researchers should implement a tiered approach to functional characterization:

• Proton conductance assays: Reconstitute purified c-subunit into liposomes loaded with pH-sensitive fluorescent dyes. Establish a pH gradient and monitor fluorescence changes that indicate proton movement through the c-ring.

• ATP synthesis coupling assays: Co-reconstitute the c-subunit with other ATP synthase components, establish a proton gradient using acid-base transition or light-driven proton pumps, and measure ATP production using luciferase-based detection systems.

• Rotational dynamics: Employ single-molecule techniques with fluorescently labeled subunits to monitor rotational movement within the assembled complex, directly correlating proton translocation with mechanical rotation.

• Inhibitor sensitivity profiles: Compare the sensitivity of reconstituted complexes to known ATP synthase inhibitors (oligomycin, venturicidin, etc.) with that of native complexes to validate functional integrity.

When designing these assays, researchers should carefully control lipid composition in reconstitution systems, as the lipid environment significantly influences ATP synthase function. For meaningful comparisons across experiments, standardized reporting of experimental conditions including protein-to-lipid ratios, buffer compositions, and temperature is essential. Additionally, incorporating positive controls using well-characterized ATP synthase components from model organisms will provide valuable reference points for interpreting results.

What approaches can resolve structural modifications in recombinant versus native atpH protein?

Resolving structural differences between recombinant and native Oedogonium cardiacum ATP synthase subunit c requires sophisticated structural biology techniques that can detect subtle modifications. Researchers should implement complementary approaches that examine structure at different resolutions:

• High-resolution mass spectrometry can identify post-translational modifications present in the native protein but absent in the recombinant form, using both top-down and bottom-up proteomics approaches.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can reveal differences in solvent accessibility and protein dynamics between native and recombinant proteins, providing insights into conformational variations.

• Circular dichroism spectroscopy allows quantitative comparison of secondary structure content, particularly important for assessing whether the recombinant protein maintains the correct alpha-helical content characteristic of c-subunits.

• NMR spectroscopy, particularly for selectively labeled proteins, can provide atomic-level information about structural differences, focusing on key functional regions like proton-binding sites.

Additionally, researchers should consider native membrane extraction techniques using styrene maleic acid lipid particles (SMALPs) to isolate native protein with its associated lipids for direct comparison with recombinant forms. Cryo-electron microscopy of both native and recombinant c-rings can reveal differences in oligomerization states or subunit arrangements. When structural differences are identified, researchers should systematically investigate whether these affect functional properties, thereby establishing structure-function relationships that inform future recombinant protein design and experimental interpretation.

How can Oedogonium cardiacum ATP synthase subunit c serve as a model for studying bioenergetic adaptation in algae?

Oedogonium cardiacum ATP synthase subunit c offers unique opportunities for studying bioenergetic adaptation in algae due to several distinctive characteristics. This protein functions within the context of a highly compact and rearranged chloroplast genome that has undergone significant evolutionary changes, including horizontal gene transfer events . Researchers can utilize this system to investigate several key aspects of bioenergetic adaptation:

• Comparative studies examining c-subunit sequence and structure across diverse algal lineages can reveal adaptive changes in proton-binding residues that may influence ATP synthesis efficiency under different environmental conditions.

• Integration of structural data with physiological measurements of photosynthetic efficiency can establish connections between molecular adaptations and organism-level energy metabolism.

• Experimental evolution approaches using Oedogonium as a model organism can track real-time adaptations in ATP synthase components under controlled selective pressures, such as fluctuating light conditions or carbon availability.

• Systems biology approaches combining transcriptomics, proteomics, and metabolomics can reveal how ATP synthase regulation is integrated with broader metabolic networks during acclimation to changing environments.

The distinct evolutionary history of Oedogonium, particularly its relationship with Stigeoclonium and divergence from the CS clade , provides a valuable comparative framework for identifying lineage-specific adaptations in bioenergetic systems. Understanding these adaptations can inform biotechnological applications, including the engineering of algal strains with enhanced bioenergetic efficiency for biofuel production or carbon capture technologies.

What research directions might exploit the unique features of Oedogonium cardiacum ATP synthase for biotechnology applications?

The unique features of Oedogonium cardiacum ATP synthase subunit c offer several promising avenues for biotechnology applications. Researchers should explore the following directions:

• Biomimetic energy conversion systems: The structural and functional characterization of this chloroplastic ATP synthase component could inform the design of artificial energy-converting membranes that mimic the efficient proton-to-ATP conversion mechanisms found in nature.

• Biosensor development: The proton-conducting properties of the c-subunit could be exploited to develop sensitive pH biosensors or proton gradient detection systems for various biotechnological applications.

• Protein engineering platforms: The recombinant expression system for this transmembrane protein could serve as a platform for testing protein engineering strategies aimed at enhancing membrane protein production or improving stability in non-native environments.

• Biophysical research tools: Well-characterized c-subunits could be developed as research tools for studying membrane protein-lipid interactions or as standards for membrane protein structural biology methods development.

• Evolutionary biomarkers: The distinctive genomic context of the atpH gene in Oedogonium could be utilized in developing molecular markers for evolutionary studies or environmental monitoring of green algal populations.

Each of these applications requires further fundamental research to fully characterize the structure-function relationships of this protein. Researchers pursuing these directions should focus on developing robust expression and purification protocols that yield consistent, high-quality protein samples suitable for downstream applications. Additionally, interdisciplinary collaborations between structural biologists, biophysicists, and biotechnology engineers will be essential for translating fundamental knowledge about this protein into practical applications.