Recombinant Idiomarina loihiensis ATP synthase subunit c (atpE)

What is Idiomarina loihiensis and why is its ATP synthase of interest?

Idiomarina loihiensis is a deep-sea gamma-proteobacterium isolated from hydrothermal vent ecosystems. The organism has evolved specialized adaptations for survival in extreme conditions, including unique membrane energetics. Its ATP synthase appears to be specific for H+ ions, indicating that unlike some other extremophiles, I. loihiensis does not rely on Na+ for membrane energetics . The organism's adaptation to the constantly changing deep-sea hydrothermal environment makes its energy-generating machinery, particularly the ATP synthase complex, of significant interest to researchers studying bioenergetics and extremophile adaptation mechanisms.

What is known about the genomic organization of ATP synthase genes in I. loihiensis?

The I. loihiensis genome has been fully sequenced, revealing its integrated mechanisms of metabolic adaptation to deep-sea hydrothermal ecosystems. Like other members of the Idiomarina genus, I. loihiensis possesses a complete set of genes encoding ATP synthase components, including the atpE gene that codes for subunit c. These genes are part of the organism's robust respiration system, with all seven examined Idiomarina strains showing 50-53 genes dedicated to respiration functions . The genes for ATP synthase components are typically organized in an operon structure similar to other gamma-proteobacteria, though their specific regulatory elements may reflect adaptations to the deep-sea environment.

How does the ATP synthase function differ in deep-sea extremophiles compared to mesophilic organisms?

ATP synthases from deep-sea extremophiles like I. loihiensis have evolved specific adaptations to function optimally under high pressure, variable temperatures, and potentially fluctuating pH conditions characteristic of hydrothermal vent environments. Unlike some extremophiles that use Na+-dependent ATP synthases, I. loihiensis maintains an H+-specific ATP synthase . This suggests that despite its extreme habitat, I. loihiensis relies on proton motive force rather than sodium motive force for ATP synthesis. The structural modifications in subunit c likely contribute to maintaining proper rotor function under these challenging conditions while preserving the fundamental mechanism of energy conversion.

What expression systems are most effective for producing recombinant I. loihiensis atpE?

Based on successful expression approaches with similar proteins, E. coli-based expression systems are generally effective for recombinant production of I. loihiensis atpE. The pET expression system, particularly pET15b vector with an N-terminal His-tag, has proven successful for similar membrane proteins from other organisms, as seen with the Clostridium tyrobutyricum phage proteins . For optimal expression of I. loihiensis atpE:

• Clone the gene into an expression vector with an appropriate tag (His-tag recommended)

• Transform into an E. coli strain optimized for membrane protein expression (C41(DE3) or C43(DE3))

• Induce expression using IPTG (0.1-1.0 mM) when cultures reach mid-log phase

• Incubate at reduced temperature (16-25°C) to promote proper folding

• Harvest cells after 4-16 hours of induction

This approach typically yields sufficient quantities of properly folded protein for subsequent purification and analysis.

What purification strategy provides the highest yield of functional recombinant atpE protein?

Purification of recombinant I. loihiensis ATP synthase subunit c requires careful consideration of its hydrophobic nature as a membrane protein. A recommended purification protocol based on similar successful approaches includes:

• Cell lysis in an appropriate buffer (20-50 mM sodium phosphate, pH 6.0-7.5, 100-300 mM NaCl)

• Membrane fraction isolation through differential centrifugation

• Solubilization using mild detergents (DDM, LDAO, or C12E8)

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

• Size exclusion chromatography as a polishing step

This approach has yielded >10 mg of partially purified protein from 250 ml cultures for similar membrane-associated proteins . Maintaining proper detergent concentrations throughout the purification process is critical for preventing protein aggregation while preserving the native conformation.

How can the structural integrity of purified recombinant atpE be verified?

To verify that purified recombinant I. loihiensis atpE maintains its structural integrity:

• SDS-PAGE analysis to confirm expected molecular weight (approximately 8-10 kDa plus tag contribution)

• Western blot analysis using antibodies against the tag or atpE-specific antibodies

• Circular dichroism (CD) spectroscopy to assess secondary structure content (expect high α-helical content)

• Size exclusion chromatography to determine oligomeric state

• Limited proteolysis to probe tertiary structure stability

• Mass spectrometry for precise molecular weight determination and identification of post-translational modifications

Functional integrity can be assessed through reconstitution into liposomes followed by proton translocation assays or ATP synthesis/hydrolysis assays when assembled with other ATP synthase components.

What techniques are most effective for studying the structure-function relationship of I. loihiensis atpE?

Several complementary approaches provide insights into the structure-function relationship of I. loihiensis atpE:

These approaches, when used in combination, can reveal how specific structural features contribute to function under extreme conditions.

How does I. loihiensis atpE compare to homologs from other extremophiles?

The ATP synthase subunit c from I. loihiensis shows interesting distinctions when compared to other extremophiles. The table below summarizes key comparative features:

This comparison reveals that while the fundamental mechanism is conserved, specific adaptations in the c-subunit enable ATP synthase function across diverse extreme environments.

What are the implications of I. loihiensis metabolism for understanding atpE function?

I. loihiensis has evolved a metabolism heavily reliant on amino acid catabolism rather than carbohydrate utilization. Genome analysis reveals that Idiomarina species have lost many genes related to carbohydrate metabolism while retaining extensive protein metabolism capabilities . The maximum number of genes in Idiomarina are found in the subsystem of amino acids and derivatives, followed by protein metabolism .

This metabolic specialization has several implications for ATP synthase subunit c function:

• The proton gradient driving ATP synthesis likely derives primarily from amino acid catabolism rather than carbohydrate oxidation

• ATP synthase must function efficiently with potentially fluctuating nutrient availability in the deep-sea environment

• The enzyme complex may be adapted to maintain optimal performance during changes in cytoplasmic pH resulting from amino acid metabolism

• Post-translational modifications of ATP synthase components might be influenced by the amino acid-rich metabolic environment

Understanding these metabolic contexts provides important insights into the physiological role and evolutionary adaptations of atpE in I. loihiensis.

What are the key considerations for designing mutation studies of I. loihiensis atpE?

When designing mutation studies of I. loihiensis atpE, researchers should consider:

• Target selection: Focus on:

  • The conserved proton-binding carboxylate residue

  • Residues unique to deep-sea adaptations (identified through multiple sequence alignment)

  • Interface residues that may affect c-ring stability under pressure

  • Residues that differ from mesophilic homologs

• Expression system optimization:

  • Use an E. coli strain optimized for membrane proteins

  • Consider codon optimization for improved expression

  • Test multiple induction conditions (temperature, IPTG concentration)

• Phenotypic assays:

  • Proton translocation efficiency

  • ATP synthesis/hydrolysis rates

  • Stability under varied pressure conditions

  • Thermal stability changes

  • Oligomeric assembly efficiency

• Controls:

  • Include wild-type I. loihiensis atpE

  • Compare with well-characterized atpE from model organisms

  • Include non-functional mutants (e.g., proton-binding site knockout)

Effective mutation studies should employ a systematic approach beginning with conserved functional residues and progressing to regions suspected to confer environmental adaptations.

How can reconstitution systems be optimized for functional studies of I. loihiensis atpE?

Optimizing reconstitution systems for functional studies of I. loihiensis atpE requires:

• Lipid composition selection:

  • Test lipid mixtures mimicking I. loihiensis native membranes

  • Consider including lipids with pressure-resistant properties

  • Adjust lipid:protein ratios (typically 50:1 to 200:1 by weight)

• Reconstitution method optimization:

  • Detergent removal by dialysis (gradual, 3-5 days)

  • Bio-Beads for controlled detergent removal

  • Direct incorporation into preformed liposomes

  • Test freeze-thaw cycles to improve incorporation

• Buffer conditions:

  • Optimize pH (typically 6.0-7.5 based on native environment)

  • Adjust salt concentration (100-300 mM NaCl)

  • Include stabilizing agents if needed (glycerol, specific lipids)

• Functional assessment:

  • Proton transport assays using pH-sensitive fluorescent dyes

  • Test function under various pressure conditions (1-400 atm)

  • Measure ATP synthesis when co-reconstituted with other ATP synthase components

Systematic optimization of these parameters will produce a reconstitution system that allows meaningful functional studies of recombinant I. loihiensis atpE under conditions relevant to its native environment.

What bioinformatic approaches can reveal evolutionary adaptations in I. loihiensis atpE?

Several bioinformatic approaches can illuminate evolutionary adaptations in I. loihiensis atpE:

• Multiple Sequence Alignment (MSA):

  • Align atpE sequences from diverse organisms including extremophiles and mesophiles

  • Identify conserved versus variable regions

  • Detect adaptive signatures in hydrophobic regions

• Phylogenetic Analysis:

  • Construct phylogenetic trees to understand evolutionary relationships

  • Compare with organismal phylogeny to identify horizontal gene transfer events

  • Use selection analysis tools (PAML, FEL, MEME) to detect positive selection

• Structural Prediction and Analysis:

  • Generate homology models based on known c-subunit structures

  • Predict stability changes under extreme conditions

  • Identify residues critical for oligomerization in the c-ring

• Comparative Genomics:

  • Analyze synteny and operon structure across Idiomarina species

  • Examine coevolution with other ATP synthase subunits

  • Compare with extremophiles from different environments

• Molecular Dynamics Simulations:

  • Predict behavior under deep-sea conditions (high pressure, varying temperature)

  • Simulate proton movement through the c-ring

  • Model conformational changes during rotation

These approaches can identify specific amino acid substitutions, structural modifications, and regulatory adaptations that enable I. loihiensis ATP synthase to function efficiently in its extreme native environment.

How can researchers address protein aggregation issues during recombinant atpE expression?

Protein aggregation is a common challenge when expressing hydrophobic membrane proteins like atpE. Researchers can employ several strategies to minimize aggregation:

• Expression system optimization:

  • Lower induction temperature (16-20°C)

  • Reduce IPTG concentration (0.1-0.5 mM)

  • Use specialized E. coli strains (C41/C43(DE3), BL21(DE3) pLysS)

  • Co-express molecular chaperones (GroEL/GroES, DnaK/DnaJ)

• Fusion partners and solubility tags:

  • N-terminal fusion with MBP, SUMO, or GST

  • C-terminal solubility enhancing peptides

  • Use cleavable tags to obtain native protein after purification

• Buffer optimization during extraction:

  • Test different extraction buffers (phosphate buffer at pH 6.0-7.0 has shown success with similar proteins)

  • Include stabilizing agents (glycerol, specific detergents)

  • Optimize salt concentration (100-300 mM NaCl)

• Detergent selection:

  • Screen multiple detergents (DDM, LDAO, C12E8, Fos-choline)

  • Test detergent mixtures for improved solubilization

  • Consider native lipid addition during solubilization

Protein extraction in 20-50 mM sodium phosphate buffer (pH 6.0-7.0) with 100-300 mM NaCl has proven effective for similar membrane proteins, yielding higher activity compared to extraction in TN buffer (20 mM Tris-HCl, pH 8, 50 mM NaCl) .

What methods can distinguish between functional and non-functional forms of recombinant atpE?

Distinguishing functional from non-functional forms of recombinant atpE requires multiple complementary approaches:

• Structural assessment:

  • Circular dichroism to confirm proper secondary structure (high α-helical content)

  • Intrinsic fluorescence to assess tertiary folding

  • Native PAGE to evaluate oligomeric assembly

• Ligand binding studies:

  • DCCD (dicyclohexylcarbodiimide) binding assay targeting the conserved carboxylate

  • Binding kinetics using isothermal titration calorimetry

  • Fluorescent probes to assess conformational states

• Functional reconstitution:

  • Proton translocation assays using pH-sensitive fluorescent dyes

  • ATP synthesis activity when co-reconstituted with other ATP synthase components

  • Patch clamp electrophysiology of reconstituted membranes

• Thermal and pressure stability:

  • Differential scanning calorimetry to measure stability

  • Activity retention after pressure treatment

  • Stability in detergent over time (size exclusion chromatography)

These approaches collectively provide a comprehensive assessment of whether recombinant atpE maintains its native functional properties or exists in a misfolded state.

How might I. loihiensis atpE be utilized in synthetic biology applications?

I. loihiensis atpE offers several promising applications in synthetic biology:

• Pressure-resistant bioenergetic systems:

  • Engineering ATP synthases for function under high pressure

  • Creating energy-generating systems for deep-sea applications

  • Developing pressure-resistant bioreactors

• Minimal ATP synthase design:

  • Using the robust nature of I. loihiensis atpE to create simplified ATP synthases

  • Engineering c-rings with altered ion specificities

  • Developing hybrid ATP synthases with enhanced stability

• Biosensors for environmental monitoring:

  • Creating pressure-responsive biosensors based on atpE conformational changes

  • Developing systems to detect environmental stressors in marine environments

  • Engineering reporter systems linked to ATP synthesis activity

• Biotechnological applications:

  • Enhancing energy production in industrial microorganisms

  • Improving protein production systems through more efficient energy generation

  • Creating robust cellular systems for extreme environments

The unique adaptations of I. loihiensis atpE to deep-sea conditions make it a valuable component for synthetic biology applications requiring robust energy-generating systems.

What emerging technologies will advance our understanding of I. loihiensis atpE structure and function?

Several emerging technologies hold promise for deeper insights into I. loihiensis atpE:

• Advanced Cryo-EM techniques:

  • Higher resolution imaging of membrane protein complexes

  • Time-resolved cryo-EM to capture different conformational states

  • In situ structural determination without extraction from native membranes

• Single-molecule studies:

  • FRET-based approaches to monitor conformational changes

  • Single-molecule force spectroscopy to measure stability

  • Direct observation of c-ring rotation under varying conditions

• Integrative structural biology:

  • Combining multiple data sources (X-ray, NMR, SAXS, modeling)

  • Multi-scale simulation approaches linking atomic movements to macroscale function

  • Artificial intelligence prediction of structure-function relationships

• High-pressure biophysical techniques:

  • High-pressure NMR and crystallography

  • Pressure-modulated activity assays

  • In situ deep-sea observations of engineered systems

• Native mass spectrometry:

  • Direct measurement of intact ATP synthase complexes

  • Determination of subunit stoichiometry

  • Identification of associated lipids and cofactors

These technologies will provide unprecedented insights into how the structural features of I. loihiensis atpE enable its function in the deep-sea environment and how these principles might be applied in biotechnology.