Recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD)

What is Idiomarina loihiensis and why is it significant in research?

Idiomarina loihiensis is a deep-sea γ-proteobacterium originally isolated from hydrothermal vents at 1,300-m depth on the Lōihi submarine volcano, Hawaii. This halophilic organism has significant research value as it survives a wide range of growth temperatures (from 4°C to 46°C) and salinities (from 0.5% to 20% NaCl) .

The organism represents a distinct lineage among γ-Proteobacteria that branched after the Pseudomonas lineage but before the Vibrio cluster. Its genome is a single circular chromosome of 2,839,318 bp with an average G+C content of 47%, containing 2,640 predicted open reading frames (ORFs), four rRNA operons, and 56 tRNA genes . The bacterium is notable for its adaptation to deep-sea hydrothermal ecosystems, primarily relying on amino acid catabolism rather than sugar fermentation for carbon and energy acquisition .

What is ATP synthase subunit beta (atpD) and what is its function in Idiomarina loihiensis?

ATP synthase subunit beta (atpD) is a critical component of the F1F0-ATP synthase complex in Idiomarina loihiensis. This protein plays a central role in energy metabolism by participating in the final step of oxidative phosphorylation, catalyzing the synthesis of ATP from ADP and inorganic phosphate using the energy of an electrochemical gradient of protons across the membrane .

The protein is characterized as part of the organism's adaptation to extreme environments, functioning efficiently in conditions of high salinity and varying temperatures. Unlike many other bacteria, I. loihiensis has evolved its ATP synthase components to function optimally in its unique ecological niche of deep-sea hydrothermal vents .

What expression systems are most effective for producing recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD)?

Current research indicates that E. coli expression systems are most commonly used for producing recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD) . This approach typically involves:

• Gene cloning into appropriate expression vectors

• Transformation into competent E. coli cells

• Induction of expression under optimized conditions

• Purification using affinity chromatography

While E. coli is the predominant host, alternative expression systems including yeast, baculovirus, and mammalian cell lines may be employed depending on research requirements . Each system offers distinct advantages:

• E. coli: High yield, cost-effective, rapid production

• Yeast: Better post-translational modifications

• Baculovirus: Improved folding of complex proteins

• Mammalian cells: Highest fidelity to native protein structure

The choice of expression system should be guided by the specific experimental needs, particularly when studying functional aspects of the protein.

What purification strategies yield the highest purity of recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD)?

Optimal purification of recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD) typically employs a multi-step approach:

• Initial capture: His-tag affinity chromatography using Ni-NTA or similar matrices is highly effective, as most recombinant versions are produced with N-terminal His tags .

• Intermediate purification: Ion exchange chromatography can remove contaminants based on charge differences.

• Polishing step: Size exclusion chromatography (gel filtration) to separate the target protein from aggregates and remaining impurities.

This strategy routinely achieves purity levels exceeding 85% as determined by SDS-PAGE . For studies requiring exceptionally high purity (>95%), additional chromatographic steps or alternative techniques such as dye-affinity chromatography may be employed, similar to purification methods used for other proteins from I. loihiensis .

How does the structure of Idiomarina loihiensis ATP synthase subunit beta (atpD) compare to other extremophile ATP synthases?

The structure of Idiomarina loihiensis ATP synthase subunit beta (atpD) shows several adaptations consistent with its extremophilic lifestyle. Comparative analysis reveals:

• Salt adaptation features: The protein likely contains a higher proportion of acidic amino acids on its surface, which is a common adaptation in halophilic proteins that helps maintain solubility and activity in high-salt environments.

• Temperature adaptations: Given the organism's ability to grow across a wide temperature range (4-46°C), the ATP synthase subunit beta likely contains structural features that provide flexibility at lower temperatures while maintaining stability at higher temperatures.

• Pressure adaptations: As a deep-sea organism from approximately 1,300m depth, the protein structure likely incorporates adaptations to withstand moderate hydrostatic pressure.

Specific structural comparisons would require detailed X-ray crystallography or cryo-EM studies, which could reveal how these adaptations manifest in the tertiary structure of the protein compared to mesophilic counterparts.

What are the enzymatic properties of recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD)?

The enzymatic properties of recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD) reflect its adaptation to extreme conditions:

• pH optimum: While specific data for the atpD subunit is not available in the search results, other enzymes from I. loihiensis such as GAPDH show optimal activity at alkaline pH (approximately 8.5) , suggesting that atpD might function optimally in slightly alkaline conditions.

• Temperature profile: The enzymatic activity likely shows a broad temperature range reflecting the organism's natural habitat, with optimal activity potentially around 45°C based on patterns observed with other I. loihiensis enzymes .

• Salt dependency: As a protein from a halophilic organism, the recombinant atpD likely requires moderate to high salt concentrations for optimal activity and stability.

• Catalytic efficiency: The protein participates in ATP synthesis as part of the F1F0-ATP synthase complex, but isolated recombinant atpD would primarily exhibit ATPase activity that can be measured through standard ATPase assays.

What are the recommended storage conditions to maintain stability of recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD)?

Based on protocols established for similar recombinant proteins from Idiomarina loihiensis, the following storage conditions are recommended for atpD:

• Short-term storage: Store at 4°C for up to one week in appropriate buffer systems .

• Long-term storage: Store at -20°C/-80°C, with -80°C preferred for extended periods. Aliquoting is necessary to avoid repeated freeze-thaw cycles .

• Storage buffer: Tris/PBS-based buffer with 6% Trehalose, pH 8.0 has been successfully used for other ATP synthase subunits from I. loihiensis .

• Reconstitution: When using lyophilized preparations, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and consider adding glycerol (5-50% final concentration) before aliquoting for storage .

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly reduces protein activity and should be strictly avoided .

How can the enzymatic activity of recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD) be accurately measured?

Several methodological approaches can be employed to accurately measure the enzymatic activity of recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD):

• ATPase activity assay: The most direct approach measures the ATP hydrolysis activity using:

  • Malachite green phosphate assay to detect released inorganic phosphate

  • Coupled enzyme assays linking ATP hydrolysis to NADH oxidation

  • Luminescent ATP detection methods similar to those used for other kinases

• Phosphorylation state analysis: Western blotting techniques using phospho-specific antibodies can detect the phosphorylation state of the protein, which is relevant to its functional status .

• Kinetic parameter determination: Full kinetic characterization should include:

  • Determination of Km and Vmax values for ATP

  • Effects of pH, temperature, and salt concentration on activity

  • Influence of various cations, particularly Mg2+, on enzymatic function

For optimal results, these assays should be performed under conditions that mimic the native environment of I. loihiensis, including appropriate salt concentrations and pH values.

How does Idiomarina loihiensis ATP synthase compare to ATP synthases from other extremophiles in terms of genomic context?

Comparative genomic analysis reveals several interesting aspects of Idiomarina loihiensis ATP synthase in relation to other extremophiles:

• Genomic organization: The ATP synthase genes in I. loihiensis are part of the core genome conserved across the Idiomarina genus, indicating their essential nature for survival in extreme environments .

• Evolutionary conservation: The ATP synthase complex is highly conserved among the seven compared genomes of Idiomarina species, with subunits being included in the 1,313 core genes identified across these species .

• Metabolic context: Unlike many bacteria that rely on carbohydrate metabolism, Idiomarina species including I. loihiensis have incomplete carbohydrate metabolic pathways. Instead, they show a higher proportion of genes involved in protein metabolism (155-190 genes) compared to carbohydrate metabolism (71-80 genes), as shown in the table below :

This suggests that ATP synthase in I. loihiensis operates in a distinctive metabolic context, primarily driven by amino acid catabolism rather than sugar metabolism .

What role does ATP synthase play in the adaptation of Idiomarina loihiensis to its extreme environment?

ATP synthase plays a critical role in I. loihiensis adaptation to extreme environments through several mechanisms:

• Energy conservation: In an environment with limited carbohydrate resources, the ATP synthase complex is crucial for efficient energy conservation, allowing the organism to maximize ATP production from the proton gradient generated by amino acid catabolism .

• Salt adaptation: The ATP synthase complex likely incorporates structural modifications that enable it to function optimally in high-salt environments, contributing to the halophilic nature of I. loihiensis .

• Temperature flexibility: The ATP synthase must maintain functionality across the wide temperature range (4-46°C) that I. loihiensis inhabits, requiring structural adaptations that balance rigidity and flexibility .

• Integration with unique metabolism: As I. loihiensis primarily relies on proteinaceous substrates rather than carbohydrates, its ATP synthase functions within a metabolic network specifically adapted for amino acid catabolism . This is evidenced by the genome encoding "various peptidases, a variety of peptide and amino acid uptake systems, and versatile signal transduction machinery" .

How can recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD) be used in structural biology studies?

Recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD) offers valuable opportunities for structural biology studies:

• X-ray crystallography: The high-purity recombinant protein (>90% as typically achieved) can be used for crystallization trials to determine its three-dimensional structure at atomic resolution.

• Cryo-electron microscopy: When combined with other ATP synthase subunits, the recombinant atpD can be used for structural determination of the entire F1F0-ATP synthase complex using cryo-EM techniques.

• Comparative structural analysis: The structure can be compared with ATP synthase subunits from mesophilic organisms to identify adaptations that confer halotolerance and thermostability.

• Structure-function relationship studies: Site-directed mutagenesis of the recombinant protein can help identify critical residues involved in catalysis, subunit interactions, or environmental adaptations.

• Protein-protein interaction studies: Techniques such as the bacterial two-hybrid assay (similar to those described in result ) can be employed to study interactions between atpD and other ATP synthase subunits or regulatory proteins.

What are the methodological considerations when studying the effect of environmental conditions on recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD) activity?

When investigating how environmental conditions affect the activity of recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD), researchers should consider:

• Buffer composition optimization:

  • Test multiple buffer systems (HEPES, Tris, phosphate) at various pH values

  • Include stabilizing agents (trehalose, glycerol) at different concentrations

  • Evaluate effects of various salt concentrations (NaCl, KCl) to mimic the native environment

• Temperature-dependent activity profiling:

  • Measure activity across a wide temperature range (4-50°C)

  • Assess thermal stability through differential scanning fluorimetry

  • Investigate cold adaptation mechanisms by comparing activity at low temperatures with mesophilic homologs

• Pressure effects assessment:

  • Consider using specialized high-pressure equipment to evaluate enzymatic activity under various hydrostatic pressures

  • Compare pressure effects on recombinant atpD with those on homologs from non-deep-sea organisms

• Experimental controls:

  • Include closely related ATP synthase subunits from non-extremophilic organisms as controls

  • Consider using multiple detection methods to confirm activity measurements

  • Validate results using both isolated subunit and reconstituted ATP synthase complex when possible

• Data analysis approaches:

  • Apply appropriate statistical methods to evaluate significance of environmental effects

  • Consider using response surface methodology to model multifactorial environmental influences

  • Develop mathematical models to predict activity under various environmental conditions

What are the major challenges in expressing and purifying functional recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD)?

Researchers commonly encounter several challenges when working with recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD):

• Protein solubility issues: As a protein from a halophilic organism, atpD may show reduced solubility in low-salt buffers commonly used during purification.

  • Solution: Optimize expression conditions using salt-supplemented media and include appropriate salt concentrations in all purification buffers.

• Protein misfolding: Expression in heterologous hosts, particularly at high rates, may lead to improper folding.

  • Solution: Consider lower induction temperatures (16-20°C), co-expression with chaperones, or exploration of alternative expression systems .

• Loss of activity during purification: The protein may lose activity during multiple purification steps.

  • Solution: Minimize processing time, maintain consistent temperature, and include stabilizing agents in all buffers.

• Tag interference with function: The commonly used His-tag may occasionally interfere with protein function.

  • Solution: Compare N-terminal and C-terminal tag placements, or consider tag removal using specific proteases after purification.

• Aggregation during storage: Purified protein may form aggregates during storage.

  • Solution: Add glycerol (5-50%) to storage buffers and strictly avoid freeze-thaw cycles .

How can researchers verify the structural integrity and functional activity of purified recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD)?

To ensure the structural integrity and functional activity of purified recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD), researchers should employ a multi-method verification approach:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure content

  • Thermal shift assays to evaluate protein stability and proper folding

  • Size exclusion chromatography to verify monodispersity and absence of aggregation

  • Limited proteolysis to assess compact folding and domain organization

• Functional verification:

  • ATPase activity assays using methods outlined in question 4.2

  • Binding assays with known interaction partners (other ATP synthase subunits)

  • Phosphorylation state analysis using techniques similar to those described for other phosphorylating enzymes

• Quality control measures:

  • SDS-PAGE and Western blotting using specific antibodies against atpD

  • Mass spectrometry to confirm protein identity and detect any post-translational modifications

  • Dynamic light scattering to assess homogeneity of the protein preparation

Complete characterization should include comparison with established benchmarks for properly folded and active ATP synthase subunits from related organisms.

What emerging research areas could benefit from studies involving recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD)?

Several cutting-edge research areas could benefit significantly from studies involving recombinant Idiomarina loihiensis ATP synthase subunit beta (atpD):

• Bioenergy applications: The ATP synthase from extremophiles like I. loihiensis could inspire the design of more robust artificial ATP-generating systems for bioenergy applications.

• Synthetic biology: Understanding the structural adaptations of atpD could inform the design of synthetic enzymes with enhanced stability in industrial conditions.

• Environmental biotechnology: The protein's adaptations to extreme conditions could provide insights for engineering enzymes capable of functioning in polluted environments or extreme industrial settings.

• Astrobiology: As a protein adapted to extreme environments, studying atpD could provide insights into potential biomolecular adaptations on other planetary bodies.

• Evolutionary biology: Detailed structural analysis could reveal evolutionary mechanisms underlying adaptation to extreme environments, particularly the deep-sea hydrothermal vent ecosystem.

• Nanotechnology: The natural molecular motor properties of ATP synthase could be harnessed for development of nanoscale devices, with the extremophile version offering enhanced stability.

How might comparative studies between Idiomarina loihiensis ATP synthase and other extremophilic ATP synthases advance our understanding of protein adaptation?

Comparative studies between I. loihiensis ATP synthase and other extremophilic ATP synthases could significantly advance our understanding of protein adaptation through:

• Identification of convergent evolution patterns: Comparing ATP synthases from unrelated extremophiles (thermophiles, psychrophiles, halophiles, barophiles) could reveal whether similar adaptive solutions have evolved independently.

• Structure-function relationship mapping: Systematic comparison of amino acid sequences, structural features, and enzymatic properties could identify specific residues and structural elements critical for adaptation to various extreme conditions.

• Molecular basis of multi-extreme adaptation: I. loihiensis is adapted to multiple extreme conditions (temperature range, salinity, depth), offering insights into how proteins reconcile potentially conflicting adaptation requirements.

• Evolutionary trajectory reconstruction: Phylogenetic analysis combined with structural comparison could reveal the evolutionary pathways that led to extremophilic adaptations in different lineages.

• Predictive model development: Data from comparative studies could be used to develop computational models predicting how proteins might adapt to specific environmental challenges, with applications in protein engineering and synthetic biology.