Recombinant Anthoceros formosae ATP synthase subunit c, chloroplastic (atpH)

What is the structural organization and functional role of ATP synthase subunit c in Anthoceros formosae?

ATP synthase subunit c in Anthoceros formosae (UniProt: P61172) is a key component of the chloroplastic F0F1-ATP synthase complex, which is essential for energy conversion in photosynthetic organisms. Structurally, this protein belongs to the ATP synthase F0 sector and forms the c-ring in the membrane-embedded portion of the complex. The c-ring functions as a rotary motor driven by proton translocation across the membrane, which ultimately powers ATP synthesis in the F1 portion of the complex .

In hornworts like Anthoceros formosae, the chloroplastic ATP synthase has evolved specific adaptations for functioning in the unique chloroplast environment. The subunit c protein contains transmembrane helices that form the proton-conducting channel. Recent structural studies of ATP synthases from various organisms have revealed that proton transfer in the lumenal half-channel typically occurs via a chain of five ordered water molecules, which likely applies to the Anthoceros formosae protein as well .

The protein is encoded by the atpH gene in the chloroplast genome and represents one of the most conserved components of the ATP synthase complex across photosynthetic lineages, reflecting its fundamental importance in energy metabolism.

What expression systems provide optimal yields for recombinant Anthoceros formosae ATP synthase subunit c?

Multiple expression systems can be employed for the production of recombinant Anthoceros formosae ATP synthase subunit c, each with distinct advantages depending on the research objectives:

The protein can be successfully purified to ≥85% purity as determined by SDS-PAGE regardless of the expression system used, though purification protocols must be optimized for each system .

What are the optimal storage and handling conditions for maintaining stability of Recombinant Anthoceros formosae ATP synthase subunit c?

Proper storage and handling of Recombinant Anthoceros formosae ATP synthase subunit c is critical for maintaining its structural integrity and functional properties:

The purified protein is optimally stored in Tris-based buffer containing 50% glycerol, which helps prevent protein denaturation during freeze-thaw cycles . The recommended storage temperature is -20°C for short-term storage (1-2 months) and -80°C for long-term preservation .

Experimental protocol considerations include:

• Avoid repeated freeze-thaw cycles, as these can lead to protein aggregation and loss of activity. Instead, prepare small working aliquots during initial thawing .

• When preparing working solutions, maintain a protein-friendly environment by including:

  • Appropriate pH buffer (typically pH 7.0-8.0)

  • Mild detergents if membrane reconstitution is required

  • Protease inhibitors to prevent degradation during experimental procedures

• For experimental manipulations, maintain the protein at 4°C whenever possible and minimize exposure to extreme pH, high salt concentrations, and oxidizing agents.

• Prior to use in functional assays, verify protein integrity via SDS-PAGE or other analytical techniques to ensure experiments are conducted with properly folded, non-degraded protein.

How does Anthoceros formosae ATP synthase subunit c contribute to oligomerization in ATP synthase complexes?

The oligomerization of ATP synthase complexes is crucial for generating membrane curvature and optimizing energy conversion efficiency. While specific data on Anthoceros formosae ATP synthase oligomerization is limited, insights can be drawn from research on related systems.

In mitochondrial ATP synthases, studies have revealed that subunit-g together with subunit-e forms an ancestral oligomerization motif shared between diverse evolutionary lineages, including trypanosomes and mammals . ATP synthase forms stable dimers that arrange into oligomeric assemblies, creating the inner-membrane curvature essential for efficient energy conversion .

For chloroplastic ATP synthases like that of Anthoceros formosae, the oligomerization mechanism may involve similar principles, with subunit c potentially participating in stabilizing interactions within the membrane domain. Experimental approaches to investigate this include:

• Cryo-electron microscopy of purified ATP synthase complexes to visualize oligomeric arrangements

• Cross-linking studies followed by mass spectrometry to identify interaction interfaces

• Site-directed mutagenesis of predicted interaction residues to disrupt oligomerization

What methodologies can be employed to study proton transport mechanisms in Anthoceros formosae ATP synthase using recombinant subunit c?

Investigating proton transport through the Anthoceros formosae ATP synthase c-ring requires specialized techniques that can detect proton movement and correlate it with structural features:

• Liposome reconstitution assays:

  • Purified recombinant subunit c can be reconstituted into liposomes containing pH-sensitive fluorescent dyes

  • Proton flux can be measured in response to artificially imposed membrane potentials

  • Data analysis should account for orientation of reconstituted proteins in the membrane

• Site-directed mutagenesis of critical residues:

  • Based on structural models, mutate conserved residues predicted to be involved in proton translocation

  • Analyze effects on proton transport rates and ATP synthesis efficiency

  • A systematic mutational approach can map the complete proton pathway

• Molecular dynamics simulations:

  • Atomistic simulations can model water molecule arrangements in the proton channel

  • Recent findings suggest that proton transfer in ATP synthases occurs via a chain of five ordered water molecules in the lumenal half-channel

  • Simulations can predict how specific amino acid residues in Anthoceros formosae subunit c coordinate these water molecules

• Hydrogen/deuterium exchange mass spectrometry:

  • This technique can identify regions of the protein that participate in proton exchange

  • Time-resolved measurements can track the kinetics of proton movement through the channel

  • Results can be correlated with functional data to develop a comprehensive model of the proton transport mechanism

The integration of these complementary approaches can provide a detailed understanding of how the unique structural features of Anthoceros formosae ATP synthase subunit c contribute to its proton transport function in chloroplastic ATP synthesis.

How can structural comparisons between Anthoceros formosae ATP synthase subunit c and homologs from other photosynthetic organisms inform evolutionary studies?

Comparative structural analysis of ATP synthase subunit c across diverse photosynthetic lineages provides valuable insights into evolutionary adaptations and functional conservation:

Methodological approach for comparative structural analysis:

• Multiple sequence alignment:

  • Align Anthoceros formosae ATP synthase subunit c (P61172) with homologs from diverse photosynthetic lineages

  • Identify conserved residues versus lineage-specific substitutions

  • Pay particular attention to transmembrane regions and residues involved in proton translocation

• Homology modeling and structural comparison:

  • Generate structural models based on available high-resolution structures

  • Compare structural features across lineages, focusing on:

    • C-ring diameter and subunit stoichiometry

    • Proton-binding sites

    • Interface regions involved in oligomerization

• Evolutionary rate analysis:

  • Calculate evolutionary rates for different protein regions

  • Identify regions under purifying selection (highly conserved) versus positive selection

  • Correlate evolutionary patterns with structural and functional domains

This comparative approach can reveal how hornworts like Anthoceros formosae have adapted their ATP synthase machinery to their unique ecological niches while maintaining the core functionality of this essential enzyme complex. It may also provide insights into the evolution of photosynthetic efficiency across plant lineages.

What protein-protein interaction assays are most effective for studying associations between Anthoceros formosae ATP synthase subunit c and other components of the ATP synthase complex?

Investigating protein-protein interactions involving Anthoceros formosae ATP synthase subunit c requires specialized approaches suitable for membrane proteins:

When studying Anthoceros formosae ATP synthase subunit c, researchers should pay particular attention to:

• Interactions within the c-ring assembly that contribute to proton channel formation

• Contacts with other F0 subunits that stabilize the membrane complex

• Dynamic interactions during rotary catalysis

• Potential interactions with the ancestral oligomerization modules (subunits e and g) that may influence ATP synthase dimerization and oligomerization

Combining multiple complementary techniques will provide the most comprehensive understanding of the interaction network involving Anthoceros formosae ATP synthase subunit c.

What are common challenges in purification of Recombinant Anthoceros formosae ATP synthase subunit c and how can they be addressed?

Purification of Recombinant Anthoceros formosae ATP synthase subunit c presents several technical challenges due to its hydrophobic nature and membrane association. Researchers typically encounter the following issues:

• Low solubility:

  • Challenge: As a membrane protein, subunit c has hydrophobic regions that can cause aggregation during extraction.

  • Solution: Use appropriate detergents (mild non-ionic detergents like DDM or LMNG) at optimized concentrations to solubilize without denaturing. Consider extraction using detergent screens to identify optimal conditions.

• Protein degradation:

  • Challenge: Proteolytic degradation during extraction and purification.

  • Solution: Maintain samples at 4°C, include protease inhibitor cocktails, minimize purification time, and verify integrity by SDS-PAGE at each purification step.

• Co-purification of contaminants:

  • Challenge: Difficulty achieving >85% purity due to co-purifying proteins.

  • Solution: Implement multi-step purification strategies, combining affinity chromatography with size exclusion and/or ion exchange techniques. Verify purity by SDS-PAGE .

• Low yield from eukaryotic expression systems:

  • Challenge: Reduced expression levels in more complex expression hosts.

  • Solution: Optimize codon usage for the expression host, evaluate different promoter systems, and consider using specialized expression strains designed for membrane proteins.

• Loss of structural integrity:

  • Challenge: Maintaining native conformation during purification.

  • Solution: Validate protein folding using circular dichroism or limited proteolysis assays before proceeding to functional studies.

Through careful optimization of these parameters, researchers can achieve the ≥85% purity necessary for meaningful biochemical and structural studies of Anthoceros formosae ATP synthase subunit c .

How can researchers distinguish between functional effects caused by mutations in Anthoceros formosae ATP synthase subunit c versus indirect structural disruptions?

When conducting site-directed mutagenesis studies on Anthoceros formosae ATP synthase subunit c, distinguishing between direct functional effects and indirect structural perturbations is crucial for accurate interpretation:

Methodological approach:

• Structural integrity assessment:

  • Circular dichroism spectroscopy to confirm secondary structure preservation in mutant proteins

  • Size exclusion chromatography to verify proper oligomeric state

  • Thermal stability assays to detect changes in protein stability

  • Limited proteolysis to probe for altered structural dynamics

• Comparative analysis framework:

  • Create a panel of mutations including:

    • Conservative substitutions (similar physicochemical properties)

    • Non-conservative substitutions

    • Known non-functional controls

  • Compare effects across this spectrum to identify structure-function relationships

• Complementary functional assays:

  • Proton transport measurements

  • ATP synthesis/hydrolysis activity

  • Membrane insertion efficiency

  • Ability to form oligomeric complexes

• Structure-guided interpretation:

  • Map mutations onto structural models

  • Consider proximity to known functional sites

  • Evaluate potential disruption of important interaction networks

  • Compare results with homologous mutations in well-studied systems

Using this comprehensive approach, researchers can confidently attribute observed functional changes to specific molecular mechanisms rather than general structural destabilization, advancing our understanding of the structure-function relationships in Anthoceros formosae ATP synthase subunit c.

What are the most informative techniques for studying the integration of Recombinant Anthoceros formosae ATP synthase subunit c into membrane environments?

Investigating membrane integration of Recombinant Anthoceros formosae ATP synthase subunit c requires specialized techniques that preserve and analyze protein-lipid interactions:

• Microscale thermophoresis (MST):

  • Enables quantitative measurement of interactions between the protein and various lipid compositions

  • Can determine binding affinities to specific lipid types found in chloroplast membranes

  • Requires minimal sample amounts and can work with membrane protein preparations

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps solvent-accessible regions versus membrane-protected domains

  • Identifies flexible regions and conformational changes upon membrane insertion

  • Provides residue-level information about membrane topology

• Atomic force microscopy (AFM) of reconstituted membranes:

  • Visualizes the organization of subunit c in lipid bilayers

  • Can detect oligomeric arrangements and structural features

  • Allows observation under near-physiological conditions

• Electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling:

  • Measures distances between strategically placed spin labels

  • Determines accessibility of specific residues to membrane vs. aqueous environments

  • Provides dynamic information about protein movement within the membrane

• Native mass spectrometry of membrane protein complexes:

  • Preserves non-covalent interactions during analysis

  • Determines stoichiometry of assembled complexes

  • Can detect bound lipids and small molecules

By combining these complementary approaches, researchers can develop a comprehensive understanding of how Anthoceros formosae ATP synthase subunit c integrates into membranes, associates with specific lipids, and assembles into functional complexes within the chloroplast environment.

How can molecular dynamics simulations enhance our understanding of Anthoceros formosae ATP synthase subunit c function?

Molecular dynamics (MD) simulations offer powerful computational approaches to study the atomic-level behavior of Anthoceros formosae ATP synthase subunit c in ways that complement experimental methods:

• Proton transport pathway mapping:

  • Simulations can identify the arrangement of water molecules in the proton channel

  • Recent research indicates that proton transfer in ATP synthases typically involves a chain of five ordered water molecules in the lumenal half-channel

  • MD can reveal how conserved residues coordinate these water molecules and facilitate proton movement

• Lipid-protein interaction analysis:

  • Simulations in explicit membrane environments can identify preferential interactions with specific lipid types

  • Can predict how the chloroplastic membrane environment influences protein stability and function

  • May reveal lipid binding sites that regulate protein activity

• Conformational dynamics visualization:

  • Microsecond-scale simulations can capture conformational changes associated with proton binding/release

  • Can identify flexible regions involved in the mechanical coupling of proton translocation to rotary motion

  • Helps interpret experimental data from structural and spectroscopic studies

• In silico mutagenesis:

  • Virtual mutations can predict functional consequences before experimental verification

  • Provides atomic-level rationale for observed phenotypes

  • Can identify residues for targeted experimental investigation

• Integration with experimental data:

These computational approaches provide mechanistic insights that would be difficult or impossible to obtain through experimental methods alone, advancing our understanding of how Anthoceros formosae ATP synthase subunit c contributes to energy conversion in photosynthetic organisms.

How does the study of Anthoceros formosae ATP synthase subunit c contribute to our understanding of chloroplast evolution?

Anthoceros formosae, as a hornwort, occupies a unique evolutionary position among photosynthetic organisms. Analysis of its ATP synthase subunit c provides valuable insights into chloroplast evolution:

Hornworts represent an early-diverging lineage of land plants with distinctive chloroplast features. The study of Anthoceros formosae ATP synthase subunit c allows researchers to trace evolutionary adaptations in energy conversion machinery across the transition from aquatic to terrestrial environments.

Key research approaches include:

• Phylogenetic analysis:

  • Comparing Anthoceros formosae ATP synthase subunit c sequences with homologs from diverse photosynthetic organisms

  • Reconstructing evolutionary history to identify conserved versus lineage-specific features

  • Evaluating selective pressures on different protein domains across evolutionary time

• Comparative genomics:

  • Analyzing atpH gene synteny and organization in the chloroplast genome

  • Examining codon usage patterns that may reflect adaptation to specific cellular environments

  • Identifying regulatory elements that control expression in different photosynthetic lineages

• Structure-function relationship across lineages:

  • Comparing residues involved in proton translocation among different photosynthetic groups

  • Identifying adaptations that optimize function in specific chloroplast environments

  • Correlating structural variations with physiological differences in ATP synthesis efficiency

This evolutionary perspective provides context for understanding how fundamental bioenergetic mechanisms have been conserved while adapting to the specific requirements of different photosynthetic strategies across plant evolution.

What can comparative studies of ATP synthase subunit c across photosynthetic organisms reveal about adaptation to different environmental conditions?

Comparative analysis of ATP synthase subunit c across diverse photosynthetic lineages offers insights into adaptive strategies for energy conversion under varying environmental conditions:

ATP synthase subunit c is present in all photosynthetic organisms from cyanobacteria to flowering plants, but has evolved specific adaptations to optimize function in different photosynthetic contexts. By comparing the Anthoceros formosae protein with homologs from other species, researchers can identify molecular adaptations that reflect environmental specialization.

Research methodologies for comparative environmental adaptation studies:

• Thermal adaptation analysis:

  • Compare ATP synthase subunit c from organisms adapted to different temperature ranges

  • Identify amino acid substitutions that confer thermal stability or flexibility

  • Correlate structural features with optimal operating temperatures

• Light intensity adaptation:

  • Examine variations in ATP synthase efficiency across species adapted to different light environments

  • Investigate how subunit c variations contribute to adjusting ATP synthesis rates to match photosynthetic electron transport capacity

  • Analyze regulatory mechanisms that coordinate ATP synthase activity with light availability

• Salinity and drought response:

  • Compare ATP synthase subunit c from organisms adapted to high-salt or low-water environments

  • Identify features that maintain function under osmotic stress

  • Examine adaptations that optimize energy conversion under resource-limited conditions

• Experimental validation approaches:

  • Heterologous expression of ATP synthase subunit c variants in model systems

  • Functional characterization under simulated environmental stress conditions

  • Site-directed mutagenesis to introduce or remove adaptive features

Through these comparative approaches, researchers can develop a broader understanding of how this essential component of the photosynthetic machinery has been fine-tuned through evolution to support life across diverse environmental niches, from aquatic environments to terrestrial ecosystems.