Recombinant Fervidobacterium nodosum ATP synthase subunit b (atpF)

What is Fervidobacterium nodosum and why is it significant for ATP synthase research?

Fervidobacterium nodosum is an obligately anaerobic, extremely thermophilic, chemoorganotrophic bacterium originally isolated from hot springs. As a member of the Thermotogae phylum, F. nodosum is characterized by its rod-shaped morphology (typically 0.5-0.6 × 1.1-2.5 µm), motility, and Gram-negative cell wall structure . The organism is particularly significant for ATP synthase research due to its remarkable temperature tolerance, with growth capability between 60-88°C (optimum around 78-80°C) and pH range of 6.5-8.5 (optimum pH 7.5) .

The thermostability of F. nodosum's proteins, including its ATP synthase components, provides a valuable model for studying protein stability mechanisms in extreme environments. The ATP synthase complex in this organism has evolved specific adaptations to maintain functionality at temperatures that would denature most mesophilic proteins, making it an excellent subject for comparative studies on thermostable enzyme engineering and energy coupling mechanisms under extreme conditions.

What is the structure and function of ATP synthase subunit b (atpF) in F. nodosum?

ATP synthase subunit b (atpF) in F. nodosum is a critical component of the F-type ATP synthase complex, specifically within the F₀ sector that spans the membrane. This protein is also referred to as ATP synthase F(0) sector subunit b, ATPase subunit I, F-type ATPase subunit b, or F-ATPase subunit b .

Functionally, the atpF protein participates in the membrane-embedded portion of ATP synthase that facilitates proton translocation across the membrane. This proton gradient drives the conformational changes in the F₁ sector that catalyze ATP synthesis from ADP and inorganic phosphate. The subunit b forms part of the peripheral stalk that connects the membrane-embedded F₀ sector with the catalytic F₁ sector, helping maintain the structural integrity of the complex during rotational catalysis .

The full-length protein consists of 161 amino acids with a molecular structure adapted to thermostability. The protein contains hydrophobic regions for membrane anchoring and more hydrophilic regions that interact with other components of the ATP synthase complex, particularly in connecting with the F₁ sector .

What are the optimal storage and handling conditions for recombinant F. nodosum atpF?

For optimal storage and handling of recombinant Fervidobacterium nodosum ATP synthase subunit b (atpF), the following conditions are recommended based on technical specifications:

Storage temperature:

• Store at -20°C for routine storage

• For extended storage periods, conserve at -20°C or -80°C

• Working aliquots can be stored at 4°C for up to one week

Buffer composition:

• Tris-based buffer containing 50% glycerol, optimized specifically for this protein

• For reconstitution, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (final concentration) is recommended for long-term storage

Handling precautions:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Repeated freezing and thawing cycles should be avoided to maintain protein integrity

• Create working aliquots to minimize freeze-thaw cycles

Shelf life:

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

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

These conditions are optimized to maintain the structural integrity and functional activity of the recombinant protein for research applications.

How can researchers verify the purity and identity of recombinant F. nodosum atpF?

Researchers can verify the purity and identity of recombinant Fervidobacterium nodosum ATP synthase subunit b (atpF) through several complementary analytical methods:

SDS-PAGE analysis:

• Commercial recombinant preparations typically guarantee >85% purity as assessed by SDS-PAGE

• The apparent molecular weight should correspond to approximately 18 kDa (161 amino acids), with possible variation depending on the expression tag used

Western blot analysis:

• Using anti-ATP synthase subunit b antibodies or anti-tag antibodies if the recombinant protein contains affinity tags

• This confirms both the identity and integrity of the protein

Mass spectrometry:

• For definitive confirmation of protein identity and sequence integrity

• MALDI-TOF or LC-MS/MS can provide peptide fingerprinting to verify the protein sequence

• This approach can also detect post-translational modifications or truncations

Functional assays:

• ATP hydrolysis assays when combined with other ATP synthase components

• Binding assays with known interaction partners such as other ATP synthase subunits

Tag verification:

• If the recombinant protein contains an affinity tag, specific assays for the tag (e.g., anti-His antibodies for His-tagged proteins)

• Note that "the tag type will be determined during the manufacturing process" for commercial preparations

When working with commercial preparations, researchers should consult the Certificate of Analysis provided by the supplier for specific quality control parameters of each lot.

What are the recommended experimental applications for recombinant F. nodosum atpF?

Recombinant Fervidobacterium nodosum ATP synthase subunit b (atpF) can be utilized in various experimental applications, leveraging its thermostable properties and role in the ATP synthase complex:

Structural studies:

• X-ray crystallography to determine high-resolution 3D structures

• Cryo-electron microscopy for visualization within the complete ATP synthase complex

• NMR spectroscopy for analyzing protein dynamics and conformational changes

Protein-protein interaction studies:

• Pull-down assays to identify interaction partners within the ATP synthase complex

• Surface plasmon resonance (SPR) to measure binding kinetics with other subunits

• Yeast two-hybrid or bacterial two-hybrid screening for novel interaction partners

• Cross-linking studies to map interaction interfaces

Thermostability investigations:

• Differential scanning calorimetry (DSC) to measure thermal unfolding transitions

• Circular dichroism (CD) spectroscopy to monitor secondary structure changes with temperature

• Comparative analyses with mesophilic homologs to identify thermostability determinants

Functional reconstitution:

• Assembly of the ATP synthase complex in liposomes to study proton translocation

• ATP synthesis/hydrolysis assays with reconstituted complexes

• Site-directed mutagenesis to identify functionally important residues

Immunological applications:

• ELISA development for detection of ATP synthase components

• Production of antibodies against the thermostable atpF protein

• Immunoprecipitation of ATP synthase complexes from thermophilic organisms

The predicted functional partners of ATP synthase components identified through protein interaction networks (such as STRING database) provide insight into potential experimental targets for interaction studies .

How can recombinant F. nodosum atpF be used for comparative studies with other organisms?

Recombinant Fervidobacterium nodosum ATP synthase subunit b (atpF) offers a valuable reference point for comparative studies across thermophilic and mesophilic organisms:

Phylogenetic analysis:

• Sequence comparison of atpF across Fervidobacterium species reveals evolutionary relationships

• F. nodosum shows varying degrees of similarity to other species: F. pennivorans (96-97%), F. islandicum (95-96%), F. changbaicum (96%), F. riparium (95%), and F. gondwanense (93%)

• Construction of phylogenetic trees to understand the evolution of thermostability in ATP synthase components

Structural comparison:

• Alignment of atpF sequences from organisms with different temperature optima

• Identification of conserved domains versus thermophile-specific adaptations

• Homology modeling to predict structural differences affecting thermostability

Functional comparative analysis:

• ATP synthesis/hydrolysis rates at different temperatures

• Comparison of pH optima and ion dependencies across species

• Assessment of protein stability under various denaturing conditions

Experimental approach for comparative studies:

• Express recombinant atpF from F. nodosum and equivalent proteins from other organisms using the same expression system

• Purify proteins under identical conditions

• Conduct parallel characterization (structural, biochemical, biophysical)

• Analyze differences in relation to the organisms' environmental adaptations

Table: Comparison of Fervidobacterium species characteristics relevant to ATP synthase studies

Such comparative approaches can reveal mechanistic insights into how ATP synthase components have adapted to function under extreme conditions, with potential applications in protein engineering for thermostability.

What expression systems are most effective for producing recombinant F. nodosum atpF?

Several expression systems have been successfully employed for producing recombinant Fervidobacterium nodosum ATP synthase subunit b (atpF), each with distinct advantages depending on research requirements:

Bacterial expression systems:

• E. coli-based expression is commonly used for recombinant atpF production

• Advantages include high yield, cost-effectiveness, and straightforward scale-up

• Considerations: potential issues with protein folding due to the thermophilic origin of the protein

• Optimization strategies: use of specialized E. coli strains (Rosetta, Arctic Express) designed for expressing proteins with rare codons or requiring lower temperature expression

Mammalian cell expression:

• Effective for producing properly folded ATP synthase components

• Advantages include post-translational modifications and improved protein solubility

• Considerations: higher cost, longer production time, and lower yield compared to bacterial systems

• Particularly useful when studying protein-protein interactions requiring mammalian-specific folding machinery

Expression strategy optimization:

• Vector selection:

  • Incorporation of appropriate promoters (T7, CMV)

  • Inclusion of affinity tags for purification (His, GST, or MBP)

  • Consideration of fusion partners to enhance solubility

• Culture conditions:

  • Temperature modulation during induction phase

  • Optimization of induction timing and concentration

  • Media composition adjustments to enhance protein expression

• Purification approach:

  • Multi-step purification including affinity chromatography

  • Ion exchange and size exclusion chromatography for higher purity

  • Typical purification yields >85% purity as assessed by SDS-PAGE

When recombinant F. nodosum atpF is expressed in heterologous systems, researchers should verify protein functionality, as the thermostable nature of this protein may affect folding and activity when expressed at lower temperatures than its native environment.

How does F. nodosum ATP synthase function at high temperatures, and what structural features contribute to its thermostability?

Fervidobacterium nodosum ATP synthase maintains functionality at extreme temperatures (up to 80°C) through several structural adaptations that contribute to its remarkable thermostability:

Key thermostability features:

• Amino acid composition:

  • Increased proportion of hydrophobic amino acids in core regions

  • Higher content of charged residues forming stabilizing salt bridges

  • Reduced number of thermolabile residues (e.g., asparagine, glutamine)

  • The atpF sequence (MDFFEINLTAVVQLLNFLFLLWILNKLLYKPFLGMMEKRKEK...) shows characteristic thermophilic adaptations with numerous hydrophobic and charged residues

• Secondary structure elements:

  • More compact α-helical and β-sheet arrangements

  • Stronger hydrogen bonding networks

  • Reduced loop regions that are susceptible to thermal denaturation

• Quaternary structure stabilization:

  • Enhanced subunit-subunit interactions in the ATP synthase complex

  • The atpF subunit interacts with other components including ATP synthase gamma subunit (atpG) with a high confidence score (0.978)

• Membrane association:

  • The hydrophobic N-terminal region of atpF anchors in the membrane, providing additional stability

  • Specialized lipid composition of F. nodosum membranes, predominantly composed of saturated fatty acids C16:0 and C18:0, contributes to membrane integrity at high temperatures

Functional adaptations:

• Proton translocation efficiency:

  • Maintained proton gradient despite increased membrane fluidity at high temperatures

  • Specialized proton channels with thermostable configurations

• Catalytic mechanism:

  • Enhanced coupling efficiency between F₀ and F₁ sectors

  • Thermostable ATP binding and hydrolysis sites

  • Modified regulatory mechanisms adapted to high-temperature environments

• Energy conservation:

  • Optimized energy coupling between proton translocation and ATP synthesis

  • Reduced proton leakage at elevated temperatures compared to mesophilic counterparts

These structural and functional adaptations collectively enable F. nodosum ATP synthase to maintain activity under conditions that would denature most proteins, making it a valuable model for understanding extreme enzyme thermostability.

What protein-protein interactions are critical for atpF function within the ATP synthase complex?

ATP synthase subunit b (atpF) participates in multiple critical protein-protein interactions that are essential for the structural integrity and functional activity of the ATP synthase complex:

Primary interaction partners:

• ATP synthase gamma subunit (atpG):

  • Interaction confidence score: 0.978 in STRING database analysis

  • Functional significance: The gamma chain regulates ATPase activity and proton flow through the complex

  • atpG "produces ATP from ADP in the presence of a proton gradient across the membrane"

• ATP synthase subunit delta (atpH):

  • Forms part of the central stalk connecting F₀ and F₁ sectors

  • The 181-amino acid atpH protein works in concert with atpF to maintain proper complex assembly

  • Critical for the regulatory functions of the ATP synthase complex

• ATP synthase subunit beta (atpD):

  • Contains catalytic sites for ATP synthesis

  • Structural association with atpF via the central and peripheral stalks

  • Complete sequence information available for interaction modeling

Interaction network:

The ATP synthase complex involves a sophisticated network of subunit interactions that collectively create the rotary engine mechanism. The atpF subunit specifically:

• Anchors to the membrane via its N-terminal domain

• Forms part of the peripheral stalk via its C-terminal domain

• Participates in the stator structure that counteracts rotation during catalysis

• Contributes to maintaining the proper distance between F₀ and F₁ sectors

Experimental evidence for interactions:

The STRING database provides evidence for functional interactions based on:

• Neighborhood: Genomic context and gene clustering

• Co-occurrence: Phylogenetic profiles

• Experimental data: Physical interaction evidence

• Database records: Curated information on protein complexes

Understanding these protein-protein interactions is critical for:

• Reconstituting functional ATP synthase complexes in vitro

• Designing rational mutations to study complex assembly and function

• Developing inhibitors or modulators of thermophilic ATP synthase activity

• Engineering thermostable ATP synthases for biotechnological applications

How can researchers effectively study the intron splicing mechanism related to atpF?

While F. nodosum atpF does not contain introns, related research on atpF introns in other organisms provides valuable methodological approaches that can be adapted for studying RNA processing in thermophilic systems:

Key methodological approaches:

• In vitro binding assays:

  • Filter binding assays can determine affinities between RNA processing factors and their targets

  • Example: CRS1 protein binding to the atpF intron showed high affinity and specificity under optimized conditions (330 mM K⁺ and 10 mM Mg²⁺)

  • Purified recombinant proteins can be used to identify direct RNA-protein interactions

• Structural mapping techniques:

  • Hydroxyl-radical footprinting to identify sites of protein-RNA interaction

  • Primer extension to map RNA structural changes upon protein binding

  • These approaches revealed that "CRS1 binding causes a shift in the structure of the intron population, such that the average structure becomes more compact"

• Functional reconstitution:

  • In vitro splicing assays to assess splicing efficiency and mechanism

  • Analysis of splicing intermediates to determine reaction pathway

  • Reconstitution of minimal splicing systems with purified components

• Comparative analysis across species:

  • Sequence comparison of atpF genes across species with varying intron structures

  • Identification of conserved versus species-specific RNA processing mechanisms

  • This approach revealed that "binding sites are not conserved in other group II introns and thus can account for CRS1's specificity for the atpF intron"

Adaptation for thermophilic systems:

• Perform assays at elevated temperatures mimicking F. nodosum's growth conditions

• Incorporate thermostable RNA processing factors from related organisms

• Optimize buffer conditions for thermostability (increased salt concentrations, stabilizing agents)

• Compare RNA processing mechanisms between thermophilic and mesophilic systems

While these methodologies were developed for studying the chloroplast atpF intron with CRS1, they provide a valuable framework for investigating RNA processing mechanisms in thermophilic systems and can be adapted for studying other aspects of RNA metabolism in F. nodosum.

What are common challenges when working with recombinant F. nodosum atpF and how can they be addressed?

Researchers working with recombinant Fervidobacterium nodosum ATP synthase subunit b (atpF) may encounter several technical challenges due to its thermophilic origin and membrane protein characteristics:

Challenge 1: Protein solubility issues

• Problem: Aggregation or inclusion body formation during expression

• Solution:

  • Use solubility-enhancing fusion tags (MBP, SUMO, or GST)

  • Express at lower temperatures (15-25°C) with extended induction times

  • Include mild detergents or lipid-like molecules in lysis buffers

  • Consider step-wise refolding protocols if retrieving protein from inclusion bodies

Challenge 2: Maintaining native conformation

• Problem: Loss of structural integrity in non-native conditions

• Solution:

  • Optimize buffer components (include glycerol at 5-50%)

  • Ensure proper ionic strength (K⁺, Mg²⁺) based on F. nodosum's native environment

  • Consider including specific lipids or lipid-like molecules

  • Store with protease inhibitors to prevent degradation

Challenge 3: Activity assessment

• Problem: Difficulty in verifying functional activity of isolated subunit

• Solution:

  • Partner with other ATP synthase components for functional reconstitution

  • Use biophysical methods (CD, DSC) to verify proper folding

  • Conduct binding assays with known interaction partners (atpG, atpH)

  • Perform thermostability assays to confirm expected thermal resistance

Challenge 4: Reproducibility between batches

• Problem: Variation in protein quality between preparations

• Solution:

  • Standardize expression and purification protocols

  • Implement multiple quality control checkpoints (SDS-PAGE, mass spectrometry)

  • Prepare larger batches and store as single-use aliquots

  • Avoid repeated freeze-thaw cycles which can cause degradation

Challenge 5: Temperature considerations

• Problem: Protein behavior differs at research lab temperatures vs. native conditions

• Solution:

  • Conduct comparative analyses at both standard and elevated temperatures

  • Consider using thermocyclers or specialized incubators for high-temperature experiments

  • Include temperature controls from mesophilic organisms for comparison

  • Document temperature-dependent behavior systematically

How can researchers verify the functional activity of recombinant F. nodosum atpF?

Verifying the functional activity of recombinant Fervidobacterium nodosum ATP synthase subunit b (atpF) requires specialized approaches since the isolated subunit does not possess enzymatic activity on its own:

Structural integrity assessment:

• Circular dichroism (CD) spectroscopy:

  • Monitors secondary structure elements

  • Confirms proper folding compared to theoretical predictions

  • Allows temperature-dependent unfolding studies to verify thermostability

• Thermal shift assays:

  • Measures protein unfolding transitions with temperature

  • Confirms expected high melting temperature characteristic of thermophilic proteins

  • Can be performed with fluorescent dyes like SYPRO Orange

Functional reconstitution approaches:

• Proteoliposome reconstitution:

  • Integrate atpF with other ATP synthase components in liposomes

  • Measure ATP synthesis driven by artificially imposed proton gradients

  • Compare activity with and without atpF to confirm its functional contribution

• Complementation assays:

  • Express F. nodosum atpF in ATP synthase-deficient strains

  • Assess restoration of oxidative phosphorylation

  • Compare with wild-type and negative controls

Interaction verification:

• Pull-down assays:

  • Use tagged recombinant atpF to capture interaction partners

  • Verify binding to other ATP synthase subunits, particularly those with high confidence interaction scores like atpG (0.978)

  • Quantify binding affinities under various conditions

• Surface plasmon resonance (SPR):

  • Measure real-time binding kinetics between atpF and partner proteins

  • Determine association and dissociation constants

  • Evaluate the effect of temperature on binding properties

Thermostability confirmation:

• Differential scanning calorimetry (DSC):

  • Directly measures thermal transitions

  • Confirms high denaturation temperature expected for a thermophilic protein

  • Provides thermodynamic parameters of unfolding

• Limited proteolysis:

  • Assess resistance to proteolytic degradation at elevated temperatures

  • Compare with mesophilic homologs

  • Identifies stable domains and flexible regions

These complementary approaches provide a comprehensive assessment of recombinant F. nodosum atpF functionality even without direct enzymatic activity measurements of the isolated subunit.

What are the key considerations for designing experiments with F. nodosum atpF in membrane-based systems?

When designing experiments with Fervidobacterium nodosum ATP synthase subunit b (atpF) in membrane-based systems, researchers should consider several critical factors to maintain protein functionality and physiological relevance:

Membrane composition considerations:

• Lipid selection:

  • Incorporate thermostable lipids similar to F. nodosum's native membrane (predominantly saturated fatty acids C16:0 and C18:0)

  • Consider using archaeal lipids or synthetic lipids with high thermal stability

  • Test different lipid compositions to optimize protein integration and function

• Membrane fluidity:

  • Account for temperature-dependent changes in membrane fluidity

  • Adjust cholesterol or equivalent components to maintain appropriate membrane properties at experimental temperatures

  • Monitor fluidity using fluorescent probes or anisotropy measurements

Experimental design parameters:

• Temperature conditions:

  • Design experiments to accommodate F. nodosum's growth temperature range (60-88°C, optimum 78-80°C)

  • Include temperature controls and gradients to assess temperature-dependent behavior

  • Ensure equipment compatibility with high-temperature experiments

• Buffer optimization:

  • Maintain pH near F. nodosum's optimum (pH 7.5)

  • Include appropriate salt concentrations (optimum 0.5 g/L NaCl)

  • Consider adding stabilizing agents like glycerol (5-50%) for prolonged experiments

• Proton gradient establishment:

  • Design methods to generate and monitor proton gradients across membranes

  • Account for higher proton permeability at elevated temperatures

  • Consider using pH-sensitive fluorescent dyes stable at high temperatures

Reconstitution methodologies:

• Proteoliposome preparation:

  • Detergent-mediated reconstitution with controlled protein:lipid ratios

  • Gentle removal of detergents via dialysis or adsorption to bio-beads

  • Verification of orientation and integration using protease protection assays

• Nanodiscs assembly:

  • Incorporation into nanodiscs for single-molecule studies

  • Selection of appropriate membrane scaffold proteins stable at higher temperatures

  • Characterization by dynamic light scattering and electron microscopy

Functional assessment:

• ATP synthesis/hydrolysis:

  • Measure activity across temperature ranges

  • Compare with mesophilic ATP synthases under identical conditions

  • Quantify the effect of proton motive force on ATP synthesis rates

• Structural integrity:

  • Monitor protein stability in membranes using fluorescence spectroscopy

  • Assess oligomeric state using cross-linking or native gel electrophoresis

  • Evaluate protein-lipid interactions using EPR or NMR spectroscopy

By carefully considering these factors, researchers can design physiologically relevant membrane-based experiments that provide insights into the functional properties of F. nodosum atpF under conditions that reflect its native thermophilic environment.