Recombinant Thermotoga sp. ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c (atpE) in Thermotoga species?

ATP synthase subunit c (atpE) in Thermotoga species is an 85 amino acid protein that forms part of the membrane-embedded c-ring of the F-type ATP synthase complex. The protein sequence (MENLGDLAQGLALLGKYLGAGLCMGIGAIGPGIGEGNIGAHAMDAMARQPEMVGTITTRMLLADA VAETTGIYSLLIAFMILLVV) reveals a predominantly hydrophobic structure suited for membrane integration .

Functionally, the c-ring participates in the rotary mechanism of ATP synthesis, working in conjunction with other subunits to convert the proton-motive force into chemical energy in the form of ATP. The c-subunit ring transfers rotary motion to the catalytic α₃β₃-headpiece through interaction with other subunits like subunit ε . In hyperthermophiles like Thermotoga species, these proteins have evolved special structural adaptations to function at extremely high temperatures (optimum around 80°C) .

How is recombinant Thermotoga sp. ATP synthase subunit c typically expressed and purified?

Recombinant Thermotoga sp. ATP synthase subunit c is typically expressed in E. coli expression systems with an N-terminal His-tag to facilitate purification . The expression vector contains the gene encoding the full-length protein (amino acids 1-85).

For purification, the following methodological approach is recommended:

• Grow transformed E. coli under appropriate induction conditions

• Harvest cells and lyse using mechanical disruption or detergent-based methods

• Perform initial purification using Ni-NTA affinity chromatography, leveraging the His-tag

• Consider a secondary purification step such as size-exclusion chromatography

• Analyze purity by SDS-PAGE (should exceed 90%)

• If necessary, concentrate the protein using centrifugal filters with appropriate molecular weight cutoffs

The purified protein is typically obtained as a lyophilized powder for long-term storage stability .

What are the optimal storage conditions for maintaining the activity of recombinant Thermotoga sp. ATP synthase subunit c?

For optimal storage of recombinant Thermotoga sp. ATP synthase subunit c, follow these evidence-based guidelines:

• Store the lyophilized powder at -20°C to -80°C upon receipt

• After reconstitution, prepare working aliquots to avoid repeated freeze-thaw cycles

• Short-term storage (up to one week) of working aliquots can be at 4°C

• For reconstituted protein requiring longer storage, add glycerol to a final concentration of 50%

• Store these glycerol-containing aliquots at -20°C or -80°C

It is particularly important to avoid repeated freeze-thaw cycles as these can significantly compromise protein integrity and activity. The recommendation for aliquoting is based on this consideration .

How does the c-subunit from Thermotoga sp. compare structurally and functionally with c-subunits from other extremophiles and mesophilic organisms?

The c-subunit from Thermotoga sp. represents an interesting model for comparative studies due to its hyperthermophilic nature. When compared to other organisms:

• Structural adaptations for thermostability:

  • Thermotoga's c-subunit likely contains a higher proportion of hydrophobic and charged residues that contribute to thermostability through enhanced hydrophobic packing and salt bridge formation

  • The amino acid sequence (MENLGDLAQGLALLGKYLGAGLCMGIGAIGPGIGEGNIGAHAMDAMARQPEMVGTITTRMLLADA VAETTGIYSLLIAFMILLVV) shows multiple glycine residues that may provide conformational flexibility needed for function at high temperatures

• Functional comparison with Mycobacterium tuberculosis:

  • While the Mtb c-ring participates in similar rotary motion mechanisms, the Thermotoga protein has evolved to function optimally at much higher temperatures

  • The c-subunit's interaction with other subunits (like ε) is structurally conserved across species but may show thermodynamic differences

• Evolutionary significance:

  • Thermotoga represents one of the deepest and most slowly evolving lineages of bacteria, making its ATP synthase components valuable for understanding the evolution of bioenergetic systems

  • The c-subunit ring structure is highly conserved across domains of life, but with adaptations specific to environmental niches

Methodologically, comparative analyses can be conducted through sequence alignment tools, homology modeling, and functional assays at different temperatures to elucidate the structural basis for thermostability.

What experimental approaches can be used to study the membrane integration and oligomerization of the Thermotoga sp. c-subunit?

Several sophisticated experimental approaches can be employed to study membrane integration and oligomerization of the Thermotoga sp. c-subunit:

• Reconstitution into liposomes or nanodiscs:

  • Reconstitute purified c-subunit into phospholipid vesicles

  • Use varying lipid compositions to assess preference for specific membrane environments

  • Monitor integration efficiency using fluorescence spectroscopy or analytical ultracentrifugation

• Cross-linking studies:

  • Apply chemical cross-linkers of varying arm lengths to identify proximity relationships

  • Use mass spectrometry to identify cross-linked peptides and infer structural organization

  • Compare cross-linking patterns under different conditions (temperature, pH) to assess structural dynamics

• Biophysical characterization of c-ring assembly:

  • Analytical ultracentrifugation to determine oligomeric state

  • Native mass spectrometry to determine precise stoichiometry

  • Single-molecule FRET to monitor assembly kinetics and subunit exchange

• Cryo-electron microscopy:

  • Visualize the c-ring structure at near-atomic resolution

  • Compare with c-rings from mesophilic organisms to identify thermostability determinants

  • Assess conformational changes under different conditions

• Functional reconstitution:

  • Assemble with other ATP synthase subunits to measure ion translocation

  • Use patch-clamp techniques to measure channel activity of reconstituted c-rings

  • Assess proton leakage similar to studies in other systems

These approaches should be calibrated for the hyperthermophilic nature of Thermotoga proteins, potentially requiring modified buffers and experimental conditions to maintain native-like environments.

How might Thermotoga sp. ATP synthase c-subunit contribute to membrane permeability and cellular bioenergetics?

The ATP synthase c-subunit may play significant roles in membrane permeability and cellular bioenergetics beyond its canonical function in ATP synthesis:

• Potential leak pathway:

  • Research has indicated that the c-subunit ring can form or contribute to leak pathways in mitochondrial membranes

  • Similar leak phenomena might occur in bacterial systems, potentially serving as a mechanism for regulating membrane potential

• Bioenergetic implications:

  • Any leak through the c-ring would affect the proton motive force and subsequently ATP synthesis efficiency

  • In Thermotoga, which lives at high temperatures, controlled proton leak might serve as a mechanism to prevent excessive membrane potential that could damage cellular components

• Metabolic regulation:

  • The link between c-subunit organization and cellular metabolism observed in other systems suggests potential regulatory roles

  • The extreme growth conditions of Thermotoga may necessitate unique regulatory mechanisms involving ATP synthase components

• Methodological approach to investigation:

  • Measure membrane potential in proteoliposomes containing purified c-subunits

  • Assess the effects of temperature, pH, and small molecule modulators on potential leak activity

  • Compare wild-type and mutant forms of the protein to identify residues critical for maintaining membrane integrity

Table 1: Potential experimental approaches to study c-subunit leak phenomena

What is the optimal protocol for reconstituting Thermotoga sp. ATP synthase subunit c for functional studies?

For optimal reconstitution of Thermotoga sp. ATP synthase subunit c, the following protocol is recommended:

• Initial preparation:

  • Briefly centrifuge the lyophilized protein vial to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% if extended storage is needed

• Membrane reconstitution for functional studies:

  • Prepare liposomes from E. coli polar lipids or synthetic phospholipids with varying acyl chain lengths suitable for high-temperature studies

  • Solubilize lipids in mild detergent (e.g., n-dodecyl-β-D-maltoside)

  • Mix purified protein with solubilized lipids at desired protein-to-lipid ratios

  • Remove detergent using Bio-Beads or dialysis

  • For hyperthermophilic proteins, gradually increase temperature during reconstitution to promote proper folding

• Verification of successful reconstitution:

  • Assess proteoliposome size distribution using dynamic light scattering

  • Confirm protein orientation using protease protection assays

  • Verify functional state using proton pumping assays with pH-sensitive fluorescent dyes

• For whole ATP synthase reconstitution:

  • Co-reconstitute with other ATP synthase subunits if studying the complete complex

  • Verify complex assembly using native gel electrophoresis

  • Assess ATP synthesis/hydrolysis activity using enzyme-coupled assays adapted for high-temperature conditions

Special consideration should be given to buffer composition, as Thermotoga proteins function optimally at elevated temperatures and may require thermostable buffer components.

What techniques can be used to measure the ion translocation activity of reconstituted Thermotoga sp. ATP synthase c-subunit?

Several specialized techniques can be employed to measure ion translocation activity of reconstituted Thermotoga sp. ATP synthase c-subunit:

• pH-sensitive fluorescent probes:

  • Incorporate probes such as ACMA (9-amino-6-chloro-2-methoxyacridine) or pyranine into proteoliposomes

  • Monitor fluorescence changes corresponding to internal pH shifts

  • Calibrate responses using ionophores like valinomycin or nigericin

  • Adapt protocol for high-temperature measurements using thermostable probes

• Ion-selective electrodes:

  • Measure bulk proton movement in real-time

  • Can be adapted for high-temperature measurements

  • Provides quantitative data on proton flux rates

• Membrane potential measurements:

  • Use potential-sensitive dyes like DiSC3(5) or Oxonol VI

  • Monitor development and dissipation of membrane potential

  • Correlate with ion movement across the membrane

• Patch-clamp electrophysiology:

  • For direct measurement of ion conductance

  • Can detect single-channel events if the c-ring forms distinct channels

  • Requires specialized equipment for high-temperature measurements

• Stopped-flow spectroscopy:

  • Measure rapid kinetics of ion movement

  • Combine with pH-sensitive or potential-sensitive probes

  • Particularly useful for temperature-dependent studies

Table 2: Comparison of ion translocation measurement techniques for Thermotoga proteins

How can researchers troubleshoot common issues when working with recombinant Thermotoga sp. ATP synthase subunit c?

Researchers working with recombinant Thermotoga sp. ATP synthase subunit c may encounter several challenges. Here are methodological approaches to troubleshoot common issues:

• Poor expression yield:

  • Optimize codon usage for E. coli expression

  • Test different E. coli strains (BL21(DE3), C41(DE3), or C43(DE3) for membrane proteins)

  • Reduce expression temperature to prevent inclusion body formation

  • Try different induction conditions (IPTG concentration, induction time)

  • Consider using specialized vectors for membrane protein expression

• Protein aggregation during purification:

  • Include appropriate detergents in all buffers (DDM, LDAO, or Triton X-100)

  • Maintain optimal salt concentration (typically 150-300 mM NaCl)

  • Add glycerol (5-10%) to stabilize protein structure

  • Perform all purification steps at 4°C despite the thermophilic nature of the protein

  • Consider on-column refolding if protein is in inclusion bodies

• Loss of activity after reconstitution:

  • Verify protein integrity by SDS-PAGE before reconstitution

  • Ensure complete detergent removal using Bio-Beads or extensive dialysis

  • Optimize lipid composition for proper membrane environment

  • Gradually transition to higher temperatures to allow proper folding

  • Test different protein-to-lipid ratios to find optimal reconstitution conditions

• Inconsistent functional assay results:

  • Standardize proteoliposome preparation (size, protein-to-lipid ratio)

  • Prepare fresh buffers and reagents for each experiment

  • Carefully control temperature during all measurements

  • Include positive and negative controls in each experiment

  • Consider the impact of freeze-thaw cycles on protein activity

• Difficulty in achieving proper oligomerization:

  • Try different detergents that preserve native oligomeric states

  • Include specific lipids that may promote c-ring assembly

  • Use gentle solubilization and purification conditions

  • Verify oligomeric state by native PAGE or analytical ultracentrifugation

By systematically addressing these issues with the suggested methodological approaches, researchers can improve the reliability and reproducibility of their experiments with this challenging but scientifically valuable protein.

How does the Thermotoga sp. ATP synthase system compare with other hyperthermophilic ATP synthases in terms of thermostability and energy coupling?

The Thermotoga sp. ATP synthase system displays several distinctive features when compared with other hyperthermophilic ATP synthases:

• Evolutionary context:

  • Thermotoga represents one of the deepest and most slowly evolving lineages of bacteria, making its ATP synthase particularly interesting from an evolutionary perspective

  • Its position near the bacterial root suggests that its ATP synthase may retain ancestral features lost in other lineages

• Thermostability mechanisms:

  • Like other hyperthermophiles, Thermotoga proteins likely employ increased hydrophobic interactions, additional salt bridges, and disulfide bonds

  • The c-subunit sequence contains multiple glycine residues that may provide the necessary flexibility while maintaining structural integrity at high temperatures

  • The specific residue composition likely reflects adaptations to function optimally at 80°C

• Energy coupling considerations:

  • Thermotoga species have unique metabolic adaptations, including the presence of both ATP-dependent and PP₁-dependent phosphofructokinases, suggesting complex energy regulation systems

  • The ATP synthase likely functions in the context of this distinctive energy metabolism

• Comparative analysis with archaeal hyperthermophiles:

  • While both use F/V-type ATP synthases, the structural details and ion specificity may differ

  • Thermotoga's ATP synthase subunits may show sequence similarities to both bacterial and archaeal counterparts, reflecting its deep evolutionary position

Table 3: Comparison of ATP synthase features across thermophilic organisms

This comparative analysis highlights the special adaptations that allow Thermotoga ATP synthase to function effectively in extreme environments while maintaining the core mechanisms of energy conversion.

What insights can be gained from studying the ATP synthase c-subunit of Thermotoga sp. for the design of thermostable biotechnological applications?

Studying the ATP synthase c-subunit from Thermotoga sp. offers valuable insights for biotechnological applications requiring thermostability:

• Design principles for thermostable membrane proteins:

  • The c-subunit's ability to maintain structure and function at 80°C provides a natural model for engineering thermostable membrane proteins

  • Specific amino acid compositions and patterns can be identified and transferred to other proteins

  • The oligomeric assembly mechanism offers insights for designing stable protein complexes

• Applications in bioenergetic systems:

  • Understanding how Thermotoga maintains efficient energy coupling at high temperatures can inform the design of artificial bioenergetic systems

  • Potential applications in biofuel cells that need to operate at elevated temperatures

  • Insights for designing proton-conducting channels with controlled leak properties

• Methodological approaches to leverage these insights:

  • Structure-guided protein engineering:

    • Identify key residues contributing to thermostability through comparative sequence analysis

    • Introduce stabilizing mutations into mesophilic counterparts

    • Verify improved stability using thermal denaturation assays

  • Chimeric protein design:

    • Create fusion proteins incorporating thermostable domains from Thermotoga

    • Test functionality and stability at elevated temperatures

    • Iteratively optimize designs based on functional outcomes

  • Reconstitution systems:

    • Develop thermostable lipid compositions inspired by Thermotoga membranes

    • Create temperature-resistant proteoliposomes for biotechnological applications

    • Engineer controlled proton/ion conductance based on c-ring principles

• Potential biotechnological applications:

  • Biosensors operating at high temperatures

  • Bioremediation processes in hot industrial effluents

  • Biocatalytic systems requiring thermostable membrane components

  • Nanoscale energy-converting devices with improved stability