Recombinant Thermosipho melanesiensis ATP synthase subunit b (atpF)

What is Thermosipho melanesiensis and why is its ATP synthase of interest to researchers?

Thermosipho melanesiensis is a thermophilic, anaerobic rod-shaped bacterium isolated from deep-sea vent hydrothermal mussels in the Lau Basin (Southwestern Pacific Ocean). It belongs to the order Thermotogales and is characterized by its distinctive outer sheath-like structure called "toga" .

This organism is particularly interesting for bioenergetic studies because:

• It grows optimally at 70°C, pH 6.5, with 30 g/L NaCl concentration

• Its doubling time under optimal conditions is approximately 100 minutes

• Its ATP synthase components have evolved to function efficiently at high temperatures

• Studying thermostable ATP synthases provides insights into mechanisms of protein stability and energy conversion in extreme environments

As a hyperthermophile, T. melanesiensis proteins like ATP synthase subunit b (atpF) offer unique structural adaptations that maintain functionality at temperatures that would denature mesophilic proteins, making them valuable for both basic research and biotechnological applications.

How should recombinant T. melanesiensis ATP synthase subunit b be reconstituted for experimental use?

Proper reconstitution of recombinant T. melanesiensis ATP synthase subunit b is critical for maintaining its structure and function. Based on established protocols, the following method is recommended :

• Initial preparation:

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

  • Carefully open the vial to prevent sample loss

• Reconstitution procedure:

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

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

  • Mix gently by inversion or mild vortexing to avoid protein denaturation

• Aliquoting:

  • Prepare small working aliquots to prevent repeated freeze-thaw cycles

  • Use low-adhesion microcentrifuge tubes to minimize protein loss

• Quality control:

  • Verify protein concentration using spectrophotometric methods

  • Confirm purity using SDS-PAGE (should be >85-90%)

The reconstituted protein can be stored at 4°C for up to one week for routine experiments. For long-term storage, keep aliquots at -20°C or preferably -80°C .

What expression systems are commonly used for producing recombinant T. melanesiensis atpF?

Several expression systems have been utilized for the production of recombinant T. melanesiensis ATP synthase subunit b, each with distinct advantages:

According to product information, commercially available recombinant T. melanesiensis atpF has been successfully expressed in both E. coli and yeast systems. The choice of expression system depends on:

• The intended experimental application

• Required purity and yield

• Need for post-translational modifications

• Resources and expertise available

For most structural and biochemical studies, E. coli-expressed protein is sufficient, while functional reconstitution experiments may benefit from yeast-expressed protein with potentially improved folding .

What storage conditions are recommended for T. melanesiensis ATP synthase subunit b to maintain its stability?

Proper storage is critical for maintaining the stability and activity of T. melanesiensis ATP synthase subunit b. Based on multiple product specifications and research protocols, the following storage recommendations are provided :

Short-term storage (up to 1 week):

• Store working aliquots at 4°C

• Avoid repeated freeze-thaw cycles

• Keep in storage buffer containing 50% glycerol when possible

Long-term storage:

• Store at -20°C for routine long-term storage

• Store at -80°C for extended storage and maximum stability

• Maintain in appropriate buffer (typically Tris-based with 50% glycerol)

Storage buffer composition:

• Tris/PBS-based buffer (pH 8.0)

• 6% Trehalose as a cryoprotectant

• 50% glycerol as stabilizing agent

Shelf life considerations:

• Liquid form: approximately 6 months at -20°C/-80°C

• Lyophilized form: approximately 12 months at -20°C/-80°C

The shelf life may vary depending on storage state, buffer ingredients, storage temperature, and the intrinsic stability of the protein itself . Repeated freeze-thaw cycles should be strictly avoided as they significantly decrease protein stability and activity.

How can researchers verify the proper folding and functionality of recombinant T. melanesiensis atpF?

Verifying proper folding and functionality of recombinant T. melanesiensis atpF requires multiple complementary approaches:

Structural integrity assessment:

• Circular dichroism (CD) spectroscopy

  • Analyze secondary structure content (α-helical content should be high for atpF)

  • Compare thermal denaturation profiles between recombinant and native proteins

  • Monitor structural stability at different temperatures (particularly at 70°C, the optimal growth temperature for T. melanesiensis)

• Size exclusion chromatography (SEC)

  • Verify monodispersity and proper oligomeric state

  • Detect potential aggregation or improper folding

• Limited proteolysis

  • Properly folded proteins show resistance to proteolytic degradation at key structural regions

  • Compare proteolytic patterns between recombinant and native proteins

Functional verification:

• Co-reconstitution assays

  • Incorporate recombinant atpF with other ATP synthase subunits

  • Evaluate complex formation by BN-PAGE (Blue Native Polyacrylamide Gel Electrophoresis)

  • Similar techniques were used to study ATP synthase complexes in other organisms

• Binding assays

  • Assess interaction with ATP synthase subunit α (atpA) and other complex components

  • Use surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC)

• ATPase activity assays in reconstituted systems

  • Similar to methods used for studying ATPase activity in T. thermophila and thermophilic bacterium PS3

  • Measure ATP hydrolysis using coupled enzyme assays at elevated temperatures

The combination of these methods provides comprehensive validation of the recombinant protein's structural integrity and functional capacity.

What methodological approaches can be used to investigate the role of atpF in ATP synthase assembly in thermophilic bacteria?

Investigating the role of atpF in ATP synthase assembly in thermophilic bacteria like T. melanesiensis requires sophisticated methodological approaches:

1. Genetic manipulation approaches:

• CRISPR-Cas9 or homologous recombination to create atpF variants

• Site-directed mutagenesis to modify key residues in the atpF sequence

• Complementation studies using atpF mutants in knockout strains

2. In vitro reconstitution experiments:

• Stepwise assembly of ATP synthase complexes with and without atpF

• Inclusion of fluorescently labeled atpF to track its incorporation into complexes

• Time-course assembly studies at different temperatures (37°C vs. 70°C)

3. Structural biology techniques:

• Cryo-electron microscopy to visualize ATP synthase assembly intermediates

• Single-particle analysis to examine structural differences in complexes with mutated atpF

• Cross-linking coupled with mass spectrometry (XL-MS) to identify interaction partners

4. Proteomic approaches:

• Similar to those used in studying Tetrahymena ATP synthase complexes

• Blue native PAGE (BN-PAGE) to resolve high molecular weight complexes

• 2D BN/BN-PAGE to identify well-resolved ATP synthase complexes

• Proteomic analysis of isolated complexes to identify interacting partners

5. Biophysical characterization:

• Analytical ultracentrifugation to study complex formation

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions involved in subunit interactions

• Thermal shift assays to determine how atpF affects complex stability at high temperatures

6. Live-cell imaging:

• Similar to techniques used to study ATP synthase trafficking in other systems

• Photoactivatable fluorescent protein tags to track assembly processes

• FRET-based approaches to monitor protein-protein interactions during assembly

These approaches provide complementary information about how atpF participates in ATP synthase assembly and how this process may be adapted to high-temperature environments.

What experimental challenges are associated with studying T. melanesiensis ATP synthase subunits, and how can they be addressed?

Studying T. melanesiensis ATP synthase subunits presents several experimental challenges that require specialized approaches:

1. Expression and purification challenges:

2. Functional reconstitution challenges:

• Challenge: Creating functional ATP synthase complexes from individual subunits

• Approach: Stepwise reconstitution protocols similar to those developed for other ATP synthases, adapting buffer conditions to mimic thermophilic environments

• Example: Reconstitution studies with T. thermophila ATP synthase demonstrated successful complex assembly using BN-PAGE analysis

3. High-temperature assay development:

• Challenge: Most standard assays are optimized for mesophilic conditions (25-37°C)

• Approach: Modify assay buffers to maintain stability at higher temperatures (60-70°C)

• Example: Studies on thermophilic bacterium PS3 ATP synthase successfully measured ATP hydrolysis at temperatures up to 60°C

4. Structural analysis challenges:

• Challenge: Obtaining high-resolution structural data for membrane proteins

• Approach: Combine cryo-EM, X-ray crystallography, and computational modeling

• Example: Single particle electron microscopy has been used effectively to study ATP synthase dimers and unique structural features in other systems

5. Simulating native membrane environment:

• Challenge: Replicating the lipid composition of T. melanesiensis membranes

• Approach: Use nanodisc technology with thermostable lipids or native membrane extraction

• Example: Studies on membrane protein transport mechanisms have successfully employed nanodiscs to maintain protein in native-like environments

6. Maintaining activity at extreme conditions:

• Challenge: Preserving enzymatic activity under thermophilic conditions during long experiments

• Approach: Develop specialized stable buffer systems with compatible detection methods

• Example: Oxygen consumption measurements in digitonin-permeabilized cells have been used to assess ATP synthase activity in thermophilic conditions

By addressing these challenges with specialized techniques, researchers can effectively study the structure, function, and adaptations of T. melanesiensis ATP synthase components.

How can researchers investigate the evolutionary adaptations of ATP synthase in extremophiles like T. melanesiensis?

Investigating evolutionary adaptations of ATP synthase in extremophiles like T. melanesiensis requires an integrated approach combining comparative genomics, structural biology, and functional analysis:

1. Comparative sequence analysis:

• Align ATP synthase subunit sequences across thermophilic, mesophilic, and psychrophilic organisms

• Identify conserved residues versus thermophile-specific substitutions

• Construct phylogenetic trees to trace evolutionary relationships

• Example finding: Comparative analysis of ATP synthase components in Thermotogales revealed lineage-specific adaptations that correlate with thermal niche

2. Structural comparison methodologies:

• Homology modeling of T. melanesiensis ATP synthase components based on known structures

• Molecular dynamics simulations at different temperatures (37°C vs. 70°C)

• Analysis of electrostatic surface potentials and hydrophobic interactions

• Similar approaches revealed unique structural features in Tetrahymena ATP synthase, including novel domains flanking c subunit rings

3. Horizontal gene transfer (HGT) analysis:

• Examine atpF and other ATP synthase genes for evidence of HGT

• Use codon usage bias and nucleotide composition analysis to identify potential foreign origin

• Example from related research: Mesotoga prima showed gene family expansion through lateral gene transfer, which could be a similar mechanism for ATP synthase adaptation

4. Chimeric protein engineering:

• Create fusion proteins combining domains from thermophilic and mesophilic ATP synthase subunits

• Test thermal stability and activity of chimeric proteins

• Identify specific regions responsible for thermostability

5. Site-directed mutagenesis experiments:

• Replace thermophile-specific amino acids with mesophilic counterparts

• Measure effects on thermal stability and catalytic efficiency

• Similar approaches have identified critical mutations in ATP synthase subunit γ that compensate for functionality in other systems

6. Integrative evolutionary analysis framework:

7. Novel subunit identification:

• Proteomic analysis of purified ATP synthase complexes to identify organism-specific subunits

• Similar approaches identified novel ATP synthase subunits in Tetrahymena that were limited to the ciliate lineage

• Investigate whether T. melanesiensis has developed unique adaptations similar to the Ymf66 protein that substitutes for subunit a in Tetrahymena

This multifaceted approach provides comprehensive insights into how ATP synthase has adapted to function efficiently in extreme environments, potentially revealing novel mechanisms of protein adaptation and evolution.

What are the methodological considerations for studying the proton translocation mechanism in thermophilic ATP synthases?

Studying proton translocation in thermophilic ATP synthases like that of T. melanesiensis presents unique methodological challenges requiring specialized techniques:

1. Membrane reconstitution systems:

• Liposome reconstitution: Use thermostable lipids (archaeal tetraether lipids) to create proteoliposomes stable at high temperatures

• Planar lipid bilayers: Establish systems capable of withstanding temperatures up to 80°C for electrophysiological measurements

• Nanodiscs: Employ thermostable membrane scaffold proteins to create nanoscale membrane environments

2. Proton flux measurement techniques:

3. Site-directed mutagenesis targets:

• Key residues: Based on the crucial buried arginines identified in other ATP synthases that form the proton channel

• Proton pathway: Mutations of potential proton-conducting residues in the membrane-spanning region of atpF

• Interface residues: Modifications at the interface between atpF and other F₀ components

4. Specialized equipment requirements:

• Temperature-controlled chambers for spectroscopic measurements

• Rapid-mixing devices resistant to high temperatures

• Thermally insulated sample holders for structural studies

5. Computational approaches:

• Molecular dynamics simulations at elevated temperatures to model proton movement

• Quantum mechanics/molecular mechanics (QM/MM) calculations to determine energetics of proton transfer

• Prediction of pKₐ shifts in key residues at high temperatures

6. Inhibitor studies:

• Test classical F₀F₁ ATP synthase inhibitors (oligomycin, DCCD) at high temperatures

• Note: Studies with T. thermophila showed unusual resistance to inhibitors like oligomycin , which might also occur in T. melanesiensis

• Develop thermostable derivatives of known inhibitors

7. Coupling ATP synthesis/hydrolysis to proton translocation:

• Measure ATP synthesis driven by artificially imposed pH gradients at high temperatures

• Conduct oxygen exchange experiments similar to those performed with thermophilic bacterium PS3

• Quantify the H⁺/ATP ratio under thermophilic conditions

These methodological considerations address the specific challenges of studying proton translocation mechanisms in thermophilic ATP synthases, enabling researchers to understand how these molecular machines maintain efficient energy conversion at extreme temperatures.

How can researchers investigate potential interactions between atpF and other ATP synthase subunits in T. melanesiensis?

Investigating the interactions between atpF and other ATP synthase subunits in T. melanesiensis requires multiple complementary approaches:

1. Crosslinking coupled with mass spectrometry (XL-MS):

• Use thermostable crosslinkers effective at 70°C (T. melanesiensis growth temperature)

• Apply mass spectrometry to identify crosslinked peptides

• Map interaction interfaces between atpF and partner subunits

• Quantify crosslinking efficiency to determine proximity relationships

2. Co-immunoprecipitation (Co-IP) assays:

• Develop antibodies against T. melanesiensis atpF or use His-tagged recombinant protein

• Perform Co-IP at physiologically relevant temperatures

• Identify interacting partners through proteomic analysis

• Similar approaches were used to confirm ATP synthase assembly in other systems

3. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC):

4. Bacterial two-hybrid (B2H) and yeast two-hybrid (Y2H) systems:

• Adapt systems for thermophilic protein interactions

• Use domain truncation to identify specific interaction regions

• Employ alanine scanning mutagenesis to identify critical residues

5. Structural biology approaches:

• Cryo-electron microscopy (cryo-EM) of the entire ATP synthase complex

• Nuclear magnetic resonance (NMR) of isolated domains

• X-ray crystallography of subunit subcomplexes

• Single-particle electron microscopy approaches similar to those used for Tetrahymena ATP synthase

6. Computational methods:

• Molecular docking of atpF with potential partner subunits

• Molecular dynamics simulations at elevated temperatures

• Coevolution analysis to identify residue pairs under coordinated selection pressure

7. FRET-based interaction analysis:

• Label atpF and partner subunits with thermostable fluorophores

• Measure FRET efficiency to determine proximity and orientation

• Perform experiments at various temperatures to assess thermal effects on interactions

8. Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

• Map protein interfaces by identifying regions protected from exchange

• Compare exchange rates in isolated subunits versus assembled complexes

• Adapt protocols for high-temperature compatibility

9. Genetic approaches:

• Construct libraries of atpF variants with mutations at potential interface residues

• Screen for assembly-deficient mutants

• Perform suppressor mutation analysis to identify compensatory changes in partner subunits

These diverse approaches provide complementary data on the interaction network between atpF and other ATP synthase components, offering insights into the molecular basis of complex assembly and stability in thermophilic environments.

What approaches can be used to study the kinetics of ATP synthesis/hydrolysis in reconstituted T. melanesiensis ATP synthase?

Studying the kinetics of ATP synthesis/hydrolysis in reconstituted T. melanesiensis ATP synthase requires specialized techniques adapted for thermophilic conditions:

1. ATP synthesis measurements:

• Luciferin-luciferase assay modified for high temperatures:

  • Use thermostable luciferase variants

  • Implement rapid sampling and immediate cooling for analysis

  • Calibrate with known ATP standards at various temperatures

• HPLC-based nucleotide quantification:

  • Direct measurement of ATP/ADP/AMP levels

  • Can be coupled with radioisotope labeling for increased sensitivity

  • Similar approaches were used to monitor carbamoyl phosphate production in thermophilic systems

• pH-jump induced ATP synthesis:

  • Create artificial proton gradients across proteoliposomes

  • Measure resulting ATP synthesis rates at temperatures up to 70°C

  • Determine the temperature dependence of ATP synthesis efficiency

2. ATP hydrolysis assays:

• Coupled enzyme assays adapted for thermophilic conditions:

  • Use thermostable coupling enzymes (pyruvate kinase, lactate dehydrogenase)

  • Monitor NADH oxidation spectrophotometrically

  • Similar approaches were used to assess ATPase activity in T. thermophila mitochondria

• Phosphate release assays:

  • Malachite green or molybdate-based colorimetric detection

  • Optimize reaction conditions for high temperature stability

  • Include appropriate controls for non-enzymatic ATP hydrolysis at elevated temperatures

• Oxygen exchange measurements:

  • Analyze 18O incorporation from water into Pi during ATP hydrolysis

  • Similar to methods used with thermophilic bacterium PS3

  • Provides insights into the catalytic mechanism and multi-site catalysis

3. Kinetic parameter determination:

4. Single-molecule approaches:

• Gold nanorod attachment to rotor subunits:

  • Visualize rotation directly using dark-field microscopy

  • Measure step size and rotation speed at different temperatures

  • Determine torque generation capabilities

• Magnetic bead rotation assays:

  • Attach magnetic beads to the rotary F1 portion

  • Apply external magnetic fields to manipulate rotation

  • Measure force-velocity relationships at elevated temperatures

5. Inhibitor studies:

• Analyze sensitivity to classical inhibitors:

  • Test oligomycin, venturicidin, and DCCD sensitivity

  • Investigate potential thermophile-specific inhibitor resistance

  • Compare with observations in other thermophilic ATP synthases

6. Reconstitution system optimization:

• Protein:lipid ratio optimization:

  • Determine optimal protein density for maximal activity

  • Test various lipid compositions mimicking thermophilic membranes

• Buffer composition effects:

  • Evaluate impact of ion concentrations on activity

  • Optimize pH for maximum stability and activity at high temperatures

These methodologies provide comprehensive insights into the kinetic properties of T. melanesiensis ATP synthase and how its catalytic mechanisms are adapted to function at high temperatures.

How does T. melanesiensis ATP synthase compare structurally and functionally to the highly divergent ATP synthase complex in Tetrahymena?

Comparing T. melanesiensis ATP synthase with the highly divergent ATP synthase complex in Tetrahymena reveals fascinating evolutionary adaptations in these distantly related organisms:

1. Subunit composition and organization:

2. Genomic encoding:

• T. melanesiensis: ATP synthase genes are primarily encoded in the bacterial genome

• Tetrahymena: Missing conventional genes for a and b subunits in the nuclear genome; uses mitochondrially encoded Ymf66 to substitute for subunit a

• Significance: Represents different evolutionary strategies for maintaining ATP synthase function

3. Structural adaptations for environment:

• T. melanesiensis: Adaptations primarily for thermostability (compact structure, increased ionic interactions)

• Tetrahymena: Adaptations for functional diversification (novel domains, unique dimer configuration)

• Shared challenge: Both organisms adapted to specialized environmental niches

4. Dimer configuration:

• Tetrahymena: ATP synthase dimers have parallel configuration rather than angled configuration seen in other organisms

• T. melanesiensis: Dimer configuration not well characterized but likely follows conventional bacterial arrangement

• Functional significance: Different approaches to membrane curvature induction and cristae formation

5. Proton translocation mechanism:

• Tetrahymena: Ymf66 (a substitute) contains buried arginines potentially forming proton channel

• T. melanesiensis: Conventional a-subunit with conserved proton pathway

• Evolutionary insight: Convergent evolution of proton translocation machinery from different protein scaffolds

6. Inhibitor sensitivity:

• Tetrahymena: Unusual resistance to classical F₀F₁ ATP synthase inhibitors (oligomycin, sodium azide)

• T. melanesiensis: Inhibitor sensitivity not well characterized but may show thermophile-specific resistance profiles

• Research opportunity: Comparative inhibitor studies could reveal mechanistic differences

7. Domain architecture:

• Tetrahymena: Contains unusually large domain (>100 kDa) on the intermembrane side and novel domains at the matrix-exposed side

• T. melanesiensis: More conventional domain architecture similar to other bacterial ATP synthases

• Evolutionary implication: Tetrahymena represents more extensive evolutionary divergence

8. ATPase activity patterns:

• Tetrahymena: Dimeric ATP synthase shows very weak ATPase activity

• T. melanesiensis: ATPase activity likely follows patterns similar to other thermophilic ATP synthases, with temperature dependence

These comparisons highlight how these distantly related organisms have evolved different solutions to the challenges of ATP synthesis, providing valuable insights into the evolutionary plasticity of this essential enzyme complex.

How can findings from T. melanesiensis ATP synthase research be applied to engineering thermostable bioenergetic systems?

Findings from T. melanesiensis ATP synthase research have significant potential for engineering thermostable bioenergetic systems with various applications:

1. Thermostable bionanomotor engineering:

• Incorporate thermostability principles from T. melanesiensis atpF into synthetic rotary motors

• Design chimeric proteins combining thermostable domains with functional domains from other organisms

• Create temperature-resistant power-generating nanomachines for high-temperature environments

2. Robust biocatalyst development:

3. Biomimetic energy conversion systems:

• Develop artificial ATP synthesis systems based on thermophilic adaptations

• Create hybrid chemical-biological energy conversion platforms operational at elevated temperatures

• Implement proton gradient-driven power generation in extreme environments

4. Thermostable membrane protein expression systems:

• Utilize T. melanesiensis-derived expression tags to enhance thermostability of heterologous membrane proteins

• Develop high-temperature protein production platforms with improved folding capabilities

• Design thermophile-based cell-free protein synthesis systems

5. Advanced biofuel cell applications:

• Engineer thermostable ATP synthase variants for high-temperature biofuel cells

• Create proton gradient-driven electrical generators inspired by thermophilic F-type ATPases

• Develop robust bio-hybrid devices for energy harvesting in extreme environments

6. Medical and biotechnological applications:

• Design thermostable protein delivery systems with enhanced storage stability

• Develop heat-resistant diagnostic enzymes based on thermophilic design principles

• Create temperature-insensitive biosensors with prolonged functional lifetimes

7. Commercial enzyme improvements:

• Apply sequence-structure-function insights to enhance stability of commercial enzymes

• Implement rational design strategies from thermophilic ATP synthase to other industrial biocatalysts

• Develop predictive models for thermostabilizing protein modifications

8. Integration with other extremophile systems:

• Combine thermophilic ATP synthase components with other extremophile-derived systems

• Create robust bioelectronic interfaces operational across wide temperature ranges

• Develop hybrid systems combining archaeal and bacterial thermophilic elements

9. Bionanotechnology platforms:

• Utilize self-assembly principles from thermophilic ATP synthase for nanoscale engineering

• Create temperature-resistant molecular machines for controlled nanomanipulation

• Develop biocompatible nanomotors with enhanced operational stability

These applications leverage the natural adaptations found in T. melanesiensis ATP synthase to create engineered systems with enhanced stability, efficiency, and functionality under extreme conditions, potentially revolutionizing bioenergetic technologies for industrial, environmental, and medical applications.