Recombinant Thermotoga neapolitana ATP synthase subunit beta (atpD)

What is Thermotoga neapolitana ATP synthase and why is it scientifically significant?

Thermotoga neapolitana ATP synthase refers to the enzyme complexes responsible for ATP synthesis or hydrolysis in this hyperthermophilic bacterium. What makes T. neapolitana particularly significant is that it possesses both F-type and V-type ATP synthases, which is the first reported instance of coexistence of both types in hyperthermophilic bacteria . This unique feature provides an excellent model system for studying:

• Molecular adaptations to extreme environments

• Evolutionary relationships between different ATP synthase types

• Specialized energy conservation mechanisms in hyperthermophiles

• Structure-function relationships in thermostable proteins

The F-type ATP synthase gene arrangement resembles those in eukaryotic organelles and bacteria, while the V-type ATP synthase has a unique gene arrangement different from those reported in archaea, bacteria, or eukaryotes .

What distinguishes F-type and V-type ATP synthases in T. neapolitana?

The following table summarizes key differences between the two ATP synthase types in T. neapolitana:

Both ATP synthase types were found to be expressed in T. neapolitana cells as confirmed by Western blot analysis, suggesting they may have specialized functional roles .

What are optimal storage conditions for recombinant T. neapolitana ATP synthase proteins?

Based on available data for recombinant T. neapolitana V-type ATP synthase subunit D, the following storage recommendations apply:

The shelf life is influenced by multiple factors including storage state, buffer composition, temperature, and the intrinsic stability of the protein . The remarkable thermostability of these proteins does not necessarily translate to enhanced storage stability at subzero temperatures.

How should researchers reconstitute lyophilized recombinant ATP synthase proteins?

For optimal reconstitution of lyophilized T. neapolitana ATP synthase proteins, follow this protocol:

• Preparation:

  • Centrifuge the vial briefly before opening to collect material at the bottom

  • Allow vial to reach room temperature to prevent condensation

• Reconstitution procedure:

  • Add deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

  • Gently mix by pipetting; avoid vigorous vortexing which may cause denaturation

  • Allow complete dissolution (5-15 minutes at room temperature)

• Long-term storage preparation:

  • Add glycerol to a final concentration of 5-50% (50% is standard)

  • Divide into working aliquots to minimize freeze-thaw cycles

  • Flash freeze aliquots and store at -20°C to -80°C

• Quality control:

  • Verify protein concentration using standard assays (Bradford, BCA)

  • Assess purity by SDS-PAGE (target >85%)

  • Optional: confirm identity by Western blot or mass spectrometry

What expression systems are effective for producing recombinant T. neapolitana ATP synthase components?

Recombinant expression of T. neapolitana ATP synthase components typically utilizes the following approaches:

For successful expression:

• Codon optimization may improve expression in E. coli

• Co-expression with chaperones can enhance proper folding

• Temperature optimization is critical for thermophilic proteins

• Inclusion of stabilizing agents (osmolytes, specific ions) may improve yield

After expression, purification typically involves cell lysis, clarification by centrifugation, and affinity chromatography (e.g., Ni-NTA for His-tagged proteins), followed by additional purification steps as needed .

What methodologies are most effective for measuring ATP synthase activity in hyperthermophilic bacteria?

Several complementary approaches can be used to assess T. neapolitana ATP synthase activity:

• Biochemical assays:

  • ATP hydrolysis assays (coupled enzyme systems)

  • Luciferase-based ATP detection methods

  • Phosphate release assays (malachite green, molybdate)

• Biophysical approaches:

  • Single-molecule rotation assays using gold nanoparticles (40-nm)

  • Fluorescence-based approaches with labeled ATP analogs

  • Membrane potential measurements using voltage-sensitive probes

• Temperature considerations:

  • Thermophilic enzymes retain significant activity at lower temperatures (e.g., 73% of maximal activity at 25°C compared to 65°C)

  • For measurements at elevated temperatures, specialized temperature-controlled chambers are required

  • Non-enzymatic ATP hydrolysis rates increase with temperature and must be accounted for

• Advanced single-molecule techniques:

  • Immobilization on NTA-modified glass surfaces via hexahistidine tags

  • Attachment of rotation probes to biotin-modified positions on rotor subunits

  • High-speed camera recording for step analysis

  • Fluctuation theorem application for torque calculations

These methodologies have revealed that thermophilic ATP synthases can generate remarkably high torque values (up to 52.4 piconewtons in some cases), which may represent an adaptation to extreme environments .

How can researchers effectively solubilize and characterize V-type ATP synthase from T. neapolitana?

The V-type ATP synthase from T. neapolitana presents significant solubilization challenges that require specialized approaches:

• Detergent screening:

  • Test a panel of detergents beyond Triton X-100 (which successfully solubilizes the F-type but not V-type ATP synthase)

  • Consider digitonin, DDM, LMNG, or amphipol alternatives

  • Evaluate detergent mixtures at various concentrations and ratios

• Buffer optimization:

  • Systematic screening of pH conditions (pH 6.0-9.0)

  • Variation of ionic strength (100-500 mM)

  • Addition of stabilizing agents:

    • Specific lipids (archaeal lipids may be particularly effective)

    • Compatible solutes (trehalose, ectoine)

    • Glycerol (10-30%)

• Alternative approaches:

  • Native lipid nanodiscs

  • Styrene-maleic acid copolymer (SMA) extraction

  • Saposin-based reconstitution

  • Cell-free expression in the presence of lipids or detergents

• Characterization strategy:

  • Initial verification by Western blotting

  • Blue native PAGE for complex integrity

  • Activity assays at various temperatures

  • Negative-stain EM followed by cryo-EM for structural characterization

The unique gene arrangement of the V-type ATPase operon in T. neapolitana suggests it may have specialized structural features requiring tailored solubilization methods .

What evolutionary insights can be gained from studying the coexistence of F-type and V-type ATPases in T. neapolitana?

The presence of both F-type and V-type ATPases in T. neapolitana offers several important evolutionary insights:

• Horizontal gene transfer implications:

  • The presence of both ATPase types suggests potential horizontal gene transfer events

  • Comparison with T. maritima, which has a complete F-type ATPase gene cluster but only a partial V-type ATPase gene (D-subunit) , indicates differential gene acquisition or loss

  • Evidence for recombination between Thermotoga lineages has been demonstrated

• Functional adaptation hypotheses:

  • Different pH optima may allow operation under varying conditions

  • Specialized coupling to different metabolic pathways

  • Adaptation to fluctuating environmental conditions in hydrothermal environments

• Comparative genomic analysis:

  • Thermotoga species differ by 3-20% in gene content despite occupying similar thermal niches

  • Genomic analysis reveals evidence of recombination between Thermotoga lineages that are sufficiently different to be considered different species

  • Sugar metabolism genes show particular variability between species, suggesting metabolic specialization

• Biogeographical considerations:

  • Thermotoga species occupy physically distinct environments in widely disparate regions of the globe

  • The distribution of specific genes (like those encoding ATP synthase components) may follow patterns distinct from species distributions

  • This has implications for how we understand microbial biogeography in extreme environments

What structural adaptations allow T. neapolitana ATP synthases to function at extreme temperatures?

T. neapolitana ATP synthases employ multiple structural strategies to maintain functionality at high temperatures:

• Primary sequence adaptations:

  • Increased proportion of charged residues forming salt bridges

  • Higher content of hydrophobic amino acids in protein cores

  • Reduction in thermolabile residues (Asn, Gln, Cys, Met)

  • Strategic placement of proline residues in loops

• Structural stabilization mechanisms:

  • Enhanced electrostatic networks throughout the protein

  • Increased number of hydrogen bonds

  • More compact folding with reduced surface-to-volume ratio

  • Optimized hydrophobic interactions in protein cores

• Functional adaptations:

  • Higher torque generation measured in some thermophilic F₁ molecules (up to 52.4 piconewtons)

  • The unitary steps coupled with ATP hydrolysis maintain the characteristic 120° pattern seen in mesophilic counterparts

  • Maintained catalytic efficiency despite structural rigidification

• Comparative performance:

  • Thermophilic ATP synthases typically retain significant activity at lower temperatures (approximately 73% of maximal activity at 25°C compared to 65°C)

  • This partial activity at moderate temperatures facilitates experimental characterization while demonstrating the broad temperature range over which these enzymes can function

These adaptations collectively contribute to the remarkable thermal stability and functionality of T. neapolitana ATP synthases in their native extreme environment.

How does the gene arrangement in T. neapolitana V-type ATPase operon differ from those in other organisms?

The gene arrangement in the T. neapolitana V-type ATPase operon has been identified as unique compared to arrangements in archaea, bacteria, and eukaryotes :

• Comparative organization:

  • Both F-type and V-type ATP synthase genes form complete operons in T. neapolitana

  • The F-type operon follows the conventional arrangement seen in bacteria and eukaryotic organelles

  • The V-type operon has a distinctive arrangement not previously reported in other organisms

• Evolutionary implications:

  • Thermotoga species show evidence of genomic recombination between lineages

  • The unique V-type ATPase operon arrangement suggests independent evolutionary history

  • Potential acquisition through horizontal gene transfer followed by rearrangement

• Functional significance:

  • Differential regulation possibilities

  • Potential co-transcription with genes not typically associated with ATP synthases

  • Possible adaptation to the hyperthermophilic lifestyle

• Research approaches to investigate these differences:

  • Comparative genomics across Thermotoga species from different geographical locations

  • Transcriptome analysis under different growth conditions

  • Promoter mapping and regulation studies

  • Functional analysis of operon structure effects on expression efficiency

How can researchers distinguish between F-type and V-type ATPase activities in T. neapolitana cell extracts?

Differentiating between the activities of the two ATP synthase types requires strategic experimental approaches:

• Selective inhibition profiles:

• Differential solubilization:

  • Treat membrane fractions with 1% Triton X-100, which selectively solubilizes F-type ATP synthase but not V-type ATP synthase

  • Measure ATP hydrolysis activity in soluble and insoluble fractions

  • Perform Western blot analysis with specific antibodies to confirm identity of enzymes in each fraction

• Biochemical separation techniques:

  • Ion exchange chromatography

  • Sucrose gradient centrifugation

  • Immunoprecipitation with subunit-specific antibodies

• Experimental workflow:

  1. Prepare membrane fractions from T. neapolitana cells

  2. Measure total ATPase activity

  3. Perform selective inhibition assays

  4. Carry out differential solubilization

  5. Identify specific subunits by immunological techniques

  6. Correlate activity with protein levels under different conditions

These approaches can help researchers attribute observed ATP hydrolysis/synthesis activities to the respective enzyme complexes and study their relative contributions to cellular energetics.