Recombinant Synechococcus elongatus ATP synthase subunit c (atpE)

What is the role of ATP synthase in Synechococcus elongatus?

ATP synthase in Synechococcus elongatus functions as the primary ATP-generating complex in photosynthesis. It plays a crucial role in energy metabolism and stress acclimation, particularly under high-light and high-temperature conditions. Recent research has demonstrated that ATP synthase is especially important for cyanobacterial stress tolerance, with mutations in specific subunits significantly affecting the organism's ability to withstand environmental challenges . The enzyme catalyzes the production of ATP from ADP and inorganic phosphate using the proton gradient established across the thylakoid membrane during photosynthesis.

How does ATP synthase in Synechococcus elongatus relate to stress response mechanisms?

ATP synthase activity directly correlates with stress tolerance in Synechococcus elongatus. Studies comparing closely related strains (S. elongatus UTEX 2973 and PCC 7942) have shown that enhanced ATP synthase activity contributes to improved tolerance to high light and high temperature . Specifically, increased ATP synthase activity leads to higher intracellular ATP levels under stress conditions, which supports protein homeostasis and photosystem II activity. This suggests that ATP synthase plays a critical role in energy-dependent stress acclimation mechanisms in cyanobacteria.

What are the optimal conditions for recombinant expression of Synechococcus elongatus ATP synthase subunits?

Based on research with other cyanobacterial proteins from Synechococcus elongatus, effective recombinant expression can be achieved using E. coli expression systems. For instance, studies with seHtpG (another protein from S. elongatus) demonstrated successful expression using both Rosetta (DE3) and BL21 (DE3)-RIL E. coli strains . Induction conditions showed flexibility, with high protein yields achieved through either 3 hours of induction at 37°C or overnight induction at 16°C following IPTG addition . For membrane proteins like AtpE, it would be prudent to consider specialized expression strains designed for membrane proteins, or to include solubilizing tags to improve expression and solubility.

What purification techniques are most effective for recombinant cyanobacterial membrane proteins?

For membrane proteins like AtpE, a multi-step purification protocol is recommended:

• Initial purification using affinity chromatography (if a His-tag or other affinity tag is included)

• Membrane solubilization using appropriate detergents (e.g., DDM, LDAO, or Triton X-100)

• Further purification by ion exchange chromatography

• Size exclusion chromatography for final polishing and to confirm oligomeric state

The choice of detergent is critical for maintaining protein stability and function. For cyanobacterial proteins, researchers have successfully used chromatography techniques to achieve high homogeneity suitable for biochemical and structural studies . It's important to validate the functional state of purified membrane proteins through activity assays to ensure the purification process hasn't compromised protein function.

What expression systems have proven most successful for producing functional AtpE?

While the search results don't specifically address AtpE expression systems, insights can be drawn from successful expression of other cyanobacterial proteins. E. coli-based expression systems have been effectively used for cyanobacterial proteins from Synechococcus elongatus, with codon-optimized strains like Rosetta (DE3) or BL21 (DE3)-RIL showing robust expression . For membrane proteins like AtpE, specialized membrane protein expression systems may be considered, including:

• C41(DE3) or C43(DE3) E. coli strains (Walker strains) designed for membrane protein expression

• Cell-free expression systems that can directly incorporate membrane proteins into nanodiscs or liposomes

• Specialized vectors containing fusion partners that enhance membrane protein expression

How can homology modeling be used to predict the structure of S. elongatus AtpE?

Homology modeling provides a valuable approach for predicting the structure of S. elongatus AtpE when experimental structures are unavailable. The methodology involves:

• Identifying suitable template structures with high sequence identity to the target protein

• Aligning the target sequence with the template structure

• Building the model using software such as MODELLER

• Energy minimization to optimize the structural model

• Validation using tools that assess structural quality

This approach has been successfully applied to AtpE from other organisms. For instance, researchers built a homology model of Mycobacterium tuberculosis AtpE using the crystal structure of AtpE from Mycobacterium phlei (PDB ID: 4V1F), which shared 84.9% sequence identity . The model was then minimized in Prime, and further analysis was performed using tools like Arpeggio to identify molecular interactions . A similar approach could be applied to S. elongatus AtpE, using available structures from closely related cyanobacteria as templates.

What techniques are available for measuring ATP synthase activity in recombinant systems?

Several techniques can be used to measure ATP synthase activity:

• ATP hydrolysis assays: Measuring phosphate release using colorimetric methods (e.g., malachite green assay)

• ATP synthesis assays: Using luciferase-based luminescence to quantify ATP production

• Proton pumping assays: Using pH-sensitive fluorescent dyes to monitor proton movement

• Membrane potential measurements: Using voltage-sensitive dyes to monitor the establishment of membrane potential

In studies with Synechococcus, researchers have successfully measured F₀F₁ ATP synthase activity in crude extracts under both normal and stress conditions . The activity can be expressed as ATP hydrolyzed per minute, with values for wild-type S. elongatus PCC 7942 and mutant strains showing measurable differences that correlate with stress tolerance .

What approaches can verify the proper folding and oligomerization of recombinant AtpE?

To verify proper folding and oligomerization of recombinant AtpE, several complementary approaches are recommended:

For AtpE specifically, which typically forms a ring structure as part of the F₀ domain, proper oligomerization is critical for function. Comparing the experimental results with known properties of native AtpE complexes is essential for validating recombinant protein quality.

How do mutations in ATP synthase subunits affect stress tolerance in Synechococcus elongatus?

Research has demonstrated that specific mutations in ATP synthase subunits can significantly impact stress tolerance in Synechococcus elongatus. A notable example is the C252Y mutation in the AtpA subunit, which confers improved tolerance to high light and high temperature stress in S. elongatus . This single amino acid substitution increases:

• AtpA protein levels under both normal and stress conditions

• Intracellular ATP synthase activity (higher F₀F₁ ATPase synthase activities)

• Intracellular ATP concentrations under stress conditions

• Photosystem II activity

• Glycogen accumulation

Site-saturation mutagenesis experiments further revealed that replacing cysteine 252 with any of four conjugated amino acids (tyrosine, tryptophan, phenylalanine, or histidine) improved stress tolerance . This suggests that specific structural modifications to ATP synthase can enhance its function under stress conditions, potentially through stabilization of the protein complex or optimization of its catalytic activity.

What computational approaches can predict the effects of mutations in AtpE?

Several computational approaches can be employed to predict the effects of mutations in AtpE:

• Stability prediction tools: Software like SDM, mCSM-Stability, and DUET can assess how mutations affect protein folding and stability

• Protein flexibility analysis: Tools such as DynaMut can predict effects on protein flexibility and conformation through normal mode analysis

• Protein-protein interaction analysis: mCSM-PPI can predict how mutations affect interactions between protein subunits

• Ligand binding analysis: mCSM-Lig can predict effects on binding affinities for substrates or inhibitors

• Evolutionary analysis: Tools like SNAP2 can provide evolutionary-based information about mutation effects

These approaches have been successfully applied to predict resistance mutations in AtpE from other organisms, such as Mycobacterium tuberculosis . Similar methodologies could be adapted for Synechococcus elongatus AtpE to guide experimental designs and interpret mutagenesis results.

How can site-directed mutagenesis be strategically designed to study AtpE function?

Strategic site-directed mutagenesis for studying AtpE function should consider:

• Conserved residues: Target highly conserved amino acids identified through multiple sequence alignments of AtpE across species

• Functional domains: Focus on residues in the proton-binding site, oligomerization interfaces, or interaction sites with other ATP synthase subunits

• Structural elements: Target residues in key structural elements like transmembrane helices or loop regions

• Comparative approach: Design mutations analogous to those that affect function in related organisms

• Rational design: Use structural models to predict which residues might impact function when mutated

The approach used for studying the C252Y mutation in AtpA provides a good model - researchers performed site-saturation mutagenesis at this position to systematically evaluate different amino acid substitutions . This comprehensive approach revealed that multiple substitutions could improve stress tolerance, providing deeper insights into structure-function relationships.

How can ATP synthase research in Synechococcus elongatus inform biotechnological applications?

Research on ATP synthase in Synechococcus elongatus has significant implications for biotechnology:

• Enhanced photosynthetic efficiency: Understanding how ATP synthase functions under various conditions can inform strategies to enhance photosynthetic efficiency in cyanobacteria and potentially in crop plants.

• Stress-tolerant strains: Identification of mutations like C252Y in AtpA that improve stress tolerance provides targets for engineering more robust cyanobacterial strains for biotechnological applications .

• Biofuel production: Optimized ATP production could enhance growth rates and biomass accumulation, improving the efficiency of biofuel production from cyanobacteria.

• Photobioreactor design: Insights into how ATP synthase performs under different conditions can inform photobioreactor design to maximize productivity.

• Protein engineering: Structural and functional insights from cyanobacterial ATP synthase can guide the engineering of more efficient ATP synthases for synthetic biology applications.

The improved stress tolerance conferred by specific ATP synthase mutations provides "a good target for future improvement of cyanobacterial stress tolerance by metabolic engineering" .

What are the current challenges in reconstituting functional ATP synthase complexes with recombinant subunits?

Reconstituting functional ATP synthase complexes presents several challenges:

• Complex assembly: ATP synthase is a multi-subunit complex, and ensuring proper assembly of all components in the correct stoichiometry is challenging.

• Membrane integration: Proper insertion of the F₀ domain (including AtpE) into membranes is critical for function but technically difficult to achieve in vitro.

• Maintaining native interactions: Preserving the precise interactions between subunits that are necessary for function is challenging in reconstituted systems.

• Post-translational modifications: Ensuring any necessary post-translational modifications are present in recombinant proteins.

• Functional assessment: Developing assays that can reliably measure the activity of reconstituted complexes under various conditions.

Solutions being explored include co-expression of multiple subunits, use of nanodiscs or liposomes for membrane protein reconstitution, and cell-free expression systems that can directly incorporate proteins into membrane environments.

How do environmental factors influence ATP synthase activity and expression in Synechococcus elongatus?

Environmental factors significantly impact ATP synthase activity and expression in Synechococcus elongatus:

• Temperature: Heat stress increases AtpA protein levels in S. elongatus, although F₀F₁ ATPase synthase activity may decrease in wild-type strains under prolonged heat stress .

• Light intensity: High light conditions affect ATP synthase activity, with strains carrying beneficial mutations (like C252Y in AtpA) maintaining higher ATP levels under high light stress .

• Nutrient availability: While not specifically addressed in the search results, nutrient conditions likely influence ATP synthase expression as part of the cell's metabolic adaptation.

• Growth phase: ATP synthase expression and activity may vary throughout the growth cycle as energy demands change.

• Circadian rhythms: As a model organism for circadian rhythm research, S. elongatus likely shows temporal regulation of ATP synthase expression.

Research has shown that strains with enhanced ATP synthase activity (through mutations like C252Y in AtpA) exhibit higher intracellular ATP concentrations under stress conditions, demonstrating the direct link between environmental conditions and ATP synthase function .

How does AtpE from Synechococcus elongatus compare with homologs from other organisms?

While specific comparative data for AtpE from Synechococcus elongatus is not provided in the search results, general principles for comparative analysis include:

• Sequence conservation: Multiple sequence alignment can reveal highly conserved regions that likely play critical functional roles.

• Structural variations: Homology modeling combined with structural alignment can identify differences in protein folding or key functional domains.

• Specialized adaptations: Unique sequence features in S. elongatus AtpE may represent adaptations to its specific ecological niche or photosynthetic lifestyle.

• Evolutionary relationships: Phylogenetic analysis can place S. elongatus AtpE in evolutionary context relative to other cyanobacteria and photosynthetic organisms.

The approach used for structural modeling of AtpE from Mycobacterium tuberculosis, which utilized homology modeling based on a related structure with high sequence identity (84.9%), provides a methodology that could be applied to comparative analysis of S. elongatus AtpE .

What can we learn from comparing ATP synthase across different Synechococcus strains?

Comparison of ATP synthase across different Synechococcus strains provides valuable insights:

• Strain-specific adaptations: The comparison between S. elongatus UTEX 2973 and PCC 7942 revealed that despite 99.8% genome identity, differences in ATP synthase (specifically the C252Y mutation in AtpA) contribute significantly to their different stress tolerance profiles .

• Structure-function relationships: Analysis of natural variations between strains helps identify which amino acid positions are important for function under different conditions.

• Evolutionary selection: Differences between closely related strains may represent evolutionary responses to specific environmental pressures.

• Performance optimization: Understanding how natural variations affect ATP synthase performance can guide protein engineering efforts.

The research comparing these closely related Synechococcus strains demonstrated that even a single amino acid substitution in ATP synthase can have profound effects on cellular physiology and stress tolerance .