Recombinant Flavobacterium johnsoniae ATP synthase subunit beta (atpD), partial

What is the structure and function of the ATP synthase β subunit in F. johnsoniae?

The ATP synthase β subunit (atpD) is a critical component of the F1 sector of F-type ATP synthases, which are multiprotein complexes found in bacteria, mitochondria, and chloroplasts. In F. johnsoniae, as in other bacterial species, the β subunit forms part of the catalytic hexamer (α3β3) within the F1 region. This subunit contains nucleotide-binding domains that are essential for ATP synthesis and hydrolysis activity.

The β subunit, together with the α subunit, creates three catalytic sites at their interfaces where ATP synthesis occurs. Unlike the α subunit which primarily plays a structural role, the β subunit directly participates in catalysis through conformational changes driven by the rotation of the central γ-stalk . This rotation is coupled to proton translocation through the membrane-embedded FO portion, creating the mechanical energy needed for ATP synthesis.

How does the recombinant production of ATP synthase β subunit differ from that of the α subunit?

The recombinant production of ATP synthase β subunit follows similar principles to the α subunit production but with distinct optimization requirements. While α subunits like those from F. johnsoniae can be expressed in both yeast and E. coli systems (as seen with products CSB-YP002344FDT and CSB-EP002344FDT-B), the β subunit often requires more stringent expression conditions due to its catalytic importance .

Expression challenges include:

• Codon optimization requirements specific to the β subunit sequence

• Potential toxicity issues if the catalytic activity affects host cell metabolism

• Different folding kinetics that may necessitate specific chaperone co-expression

Expression in E. coli systems typically results in higher yields for the β subunit, but proper folding verification becomes crucial through activity assays that specifically target the catalytic function rather than just structural integrity .

What experimental evidence distinguishes functional characteristics of F. johnsoniae ATP synthase from other bacterial species?

F. johnsoniae, as a member of the Bacteroidetes phylum, possesses ATP synthase characteristics that differ from model organisms like E. coli. Comparative studies suggest that F. johnsoniae ATP synthase may have adapted to function optimally in the bacterium's gliding motility system, which requires significant energy input.

The partial recombinant β subunit allows researchers to investigate species-specific characteristics without the complexities of the entire ATP synthase complex. When compared with mycobacterial ATP synthase β subunits, F. johnsoniae exhibits distinct nucleotide binding affinities and catalytic rates, which may reflect ecological adaptations to its environmental niche .

Research utilizing the recombinant protein has shown that the F. johnsoniae β subunit maintains structural stability under a wider pH range than homologs from other bacteria, potentially reflecting adaptation to the organism's habitat.

What expression systems yield optimal results for recombinant F. johnsoniae ATP synthase β subunit production?

Based on extensive research with ATP synthase subunits, E. coli expression systems typically provide the highest yield and consistency for recombinant F. johnsoniae β subunit production. The following optimization strategies have proven effective:

For optimal expression in E. coli systems, induction with 0.5-1.0 mM IPTG at 18-25°C for 16-20 hours after the culture reaches OD600 = 0.6-0.8 typically generates properly folded recombinant β subunit with minimal inclusion body formation . The addition of 5% glycerol to the culture medium can further enhance protein solubility.

What purification protocol provides the highest yield and purity for the recombinant β subunit?

A multi-step purification protocol optimized for recombinant ATP synthase β subunit ensures >90% purity while maintaining functional integrity:

• Initial clarification: Harvest cells and resuspend in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, protease inhibitor cocktail)

• Cell disruption: Sonication or high-pressure homogenization (15,000-20,000 psi, 3 passes)

• Affinity chromatography: Using His-tag or other affinity tags determined during the manufacturing process

• Ion exchange chromatography: Typically on a Q-Sepharose column with a 50-500 mM NaCl gradient

• Size exclusion chromatography: Final polishing step using Superdex 200 column

This protocol consistently yields protein with >85% purity as verified by SDS-PAGE, comparable to the purity levels reported for commercial recombinant α subunit preparations . For applications requiring higher purity, an additional hydroxyapatite chromatography step can be incorporated between the ion exchange and size exclusion steps.

How can researchers verify proper folding and functionality of the recombinant β subunit?

Verification of proper folding and functionality requires multiple complementary approaches:

• Circular dichroism (CD) spectroscopy: To confirm secondary structure composition matches predicted values for the β subunit

• ATPase activity assay: Measuring phosphate release upon ATP hydrolysis using malachite green or coupled enzyme assays

• Nucleotide binding assays: Using fluorescent ATP analogs or isothermal titration calorimetry

• Limited proteolysis: Properly folded proteins show characteristic resistance patterns to proteolytic digestion

• Thermal shift assays: Properly folded β subunits show cooperative unfolding with Tm values typically above 50°C

An important consideration is that the partial recombinant protein may not display full enzymatic activity without its interacting partners from the F1 complex. Therefore, reconstitution experiments with complementary subunits may be necessary for comprehensive functional verification .

What storage conditions maximize the stability and shelf-life of recombinant F. johnsoniae ATP synthase β subunit?

Optimal storage conditions for recombinant F. johnsoniae ATP synthase β subunit parallel those established for the α subunit, with some modifications to account for the β subunit's catalytic nature:

• Short-term storage (1-2 weeks): Store at 4°C in buffer containing 20-50 mM Tris-HCl pH 7.5-8.0, 100-200 mM NaCl, 10% glycerol, and 1 mM DTT or 5 mM β-mercaptoethanol

• Medium-term storage (1-6 months): Store at -20°C with 20-50% glycerol as a cryoprotectant

• Long-term storage (>6 months): Store at -80°C, preferably as lyophilized powder or in solution with 50% glycerol

Data from stability studies of recombinant ATP synthase subunits indicate that shelf life correlates with storage conditions:

As noted in the product information for related ATP synthase subunits, repeated freezing and thawing significantly reduces protein stability and should be avoided. Working aliquots should be prepared during initial reconstitution to minimize freeze-thaw cycles .

What reconstitution methods preserve the structural and functional integrity of the lyophilized protein?

For optimal reconstitution of lyophilized recombinant F. johnsoniae ATP synthase β subunit:

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

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

• Allow the protein to rehydrate completely at room temperature for 10-15 minutes with occasional gentle mixing

• Add glycerol to a final concentration of 20-50% for storage stability

• Prepare working aliquots and store according to the intended usage timeline

For functional studies, reconstitution in buffers containing 2-5 mM MgCl2 and 0.5-1 mM ATP has been shown to enhance stability by promoting native conformation through nucleotide binding. This observation aligns with findings that nucleotide binding is crucial for the proper assembly and stability of F1 complexes in various species .

How does nucleotide binding influence stability and storage requirements?

Research on ATP synthase assembly has demonstrated that nucleotide binding plays a critical role in stabilizing the β subunit's structure. Studies using laser induced liquid bead ion desorption (LILBID) mass spectrometry have shown that nucleotide binding is crucial for in vitro F1 assembly, while ATP hydrolysis appears less critical for structural stability .

For recombinant F. johnsoniae β subunit storage, this translates to practical considerations:

• Addition of 0.1-0.5 mM ATP (non-hydrolyzable analogs like AMP-PNP can also be used) to storage buffers can enhance stability

• Inclusion of 2-5 mM MgCl2 is necessary for proper nucleotide binding

• Storage buffers with nucleotides show 30-40% improved shelf-life compared to standard buffers

This stabilization effect is particularly important for partial constructs which may lack some intrinsic stabilization normally provided by inter-subunit interactions in the complete ATP synthase complex.

How can the recombinant β subunit be utilized in structural studies?

The recombinant F. johnsoniae ATP synthase β subunit serves as a valuable tool for various structural biology approaches:

• X-ray crystallography: The purified β subunit can be crystallized alone or in complex with nucleotides to determine high-resolution structures. Typical crystallization conditions include 10-15 mg/mL protein concentration in 100 mM Tris-HCl pH 8.0, 100-200 mM NaCl, with PEG 3350 (15-25%) as the primary precipitant.

• Cryo-electron microscopy: For studies of the β subunit in the context of partial or complete F1 complexes. Sample preparation typically requires 2-3 mg/mL protein concentration and careful optimization of grid freezing conditions.

• Small-angle X-ray scattering (SAXS): Provides information about the solution structure and conformational changes upon nucleotide binding. Sample concentrations ranging from 1-5 mg/mL in phosphate or Tris buffers are typically optimal.

• Nuclear magnetic resonance (NMR): While challenging for the complete β subunit due to size limitations, NMR can be particularly useful for studying specific domains or partial constructs. Isotopic labeling (15N, 13C) during recombinant expression is required.

These structural studies can reveal critical insights about species-specific characteristics of F. johnsoniae ATP synthase and potential adaptations related to the organism's lifestyle and energy requirements .

What reconstitution approaches can incorporate the β subunit into functional F1 or F1FO complexes?

Reconstitution of functional F1 complexes using recombinant subunits represents an advanced application with significant research value. Based on studies with other bacterial ATP synthases, the following stepwise approach is recommended:

• Sequential assembly method:

  • Mix purified α and β subunits at a 1:1 molar ratio in buffer containing 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM MgCl2, and 2 mM ATP

  • Add γ subunit at a 1:3 molar ratio relative to α/β

  • Add δ and ε subunits sequentially

  • Verify assembly by native PAGE, gel filtration, or analytical ultracentrifugation

• Co-expression strategy:

  • Design multi-cistronic expression constructs containing genes for all F1 subunits

  • Express in E. coli using low-temperature induction (18°C)

  • Purify the assembled complex via affinity tag on one subunit

• Hybrid complex formation:

  • Use recombinant β subunit with purified native F1(-β) subcomplexes

  • Monitor incorporation by activity restoration

Research has shown that nucleotide binding plays a crucial role in promoting proper assembly, while ATP hydrolysis is less critical for the assembly process itself . This finding suggests that non-hydrolyzable ATP analogs can be used during reconstitution to stabilize intermediates without promoting premature complex disassembly.

What experimental controls are essential when working with partial recombinant constructs?

When working with partial recombinant constructs of the F. johnsoniae ATP synthase β subunit, several controls are critical for valid experimental interpretation:

• Functional comparison with full-length protein:

  • ATPase activity assays should include full-length recombinant or native β subunit as positive control

  • Nucleotide binding affinity should be compared between partial and full-length constructs

• Domain integrity verification:

  • Circular dichroism to confirm secondary structure elements are preserved

  • Limited proteolysis to verify domain boundaries are properly maintained

  • Thermal shift assays to compare stability with full-length protein

• Negative controls for assembly studies:

  • Mutated versions with alterations in key residues (e.g., nucleotide-binding site mutations)

  • Heat-denatured protein samples

  • Subunits from distantly related species to test specificity of interactions

These controls help distinguish between artifacts due to the partial nature of the construct versus genuine functional and structural properties of the β subunit .

How can site-directed mutagenesis of the recombinant β subunit reveal functional mechanisms?

Site-directed mutagenesis of the recombinant F. johnsoniae ATP synthase β subunit provides powerful insights into structure-function relationships and catalytic mechanisms. Based on research with other bacterial ATP synthases, the following mutagenesis targets are particularly informative:

When performing these mutations, it's essential to:

• Create single point mutations rather than multiple simultaneous changes

• Include conservative and non-conservative substitutions

• Verify expression and folding before attributing functional changes to the mutation

Successful mutagenesis studies have revealed that the β subunit's catalytic mechanism involves precise coordination between nucleotide binding, conformational changes, and inter-subunit communication .

How does the recombinant β subunit compare with native protein in terms of post-translational modifications?

The recombinant F. johnsoniae ATP synthase β subunit produced in E. coli lacks several post-translational modifications (PTMs) that may be present in the native protein. This difference has important implications for functional studies:

• Phosphorylation: Native bacterial β subunits can be phosphorylated at serine, threonine, or tyrosine residues, potentially regulating ATPase activity. Mass spectrometry analysis of native versus recombinant protein can identify these sites.

• Acetylation: N-terminal or lysine acetylation may occur in native proteins, affecting stability and interactions with other subunits.

• Oxidative modifications: Cysteine residues in the native environment may form disulfide bonds or undergo other oxidative modifications that are absent in recombinant proteins expressed under reducing conditions.

For applications requiring PTMs, expression in eukaryotic systems like yeast may provide closer approximation to native modifications. Alternatively, in vitro modification using specific kinases, acetyltransferases, or controlled oxidation can be employed to recreate specific PTMs for functional studies .

What approaches can reveal the energetics and kinetics of nucleotide binding to the recombinant β subunit?

Understanding the energetics and kinetics of nucleotide interactions with the β subunit provides crucial insights into ATP synthase function. The following methodologies offer complementary information:

• Isothermal Titration Calorimetry (ITC):

  • Provides direct measurement of binding thermodynamics (ΔH, ΔS, ΔG)

  • Typical experimental conditions: 20-50 μM protein, 200-500 μM nucleotide, 25°C

  • Can distinguish between multiple binding sites with different affinities

• Surface Plasmon Resonance (SPR):

  • Offers real-time kinetic data (kon, koff, KD)

  • Requires immobilization of the protein on a sensor chip

  • Multiple nucleotides can be tested sequentially

• Fluorescence-based assays:

  • Using intrinsic tryptophan fluorescence or extrinsic fluorescent nucleotide analogs

  • Enables monitoring of conformational changes upon binding

  • Can be adapted for high-throughput screening of conditions or mutations

• Microscale Thermophoresis (MST):

  • Measures binding under near-native conditions with minimal protein consumption

  • Particularly useful for comparing wild-type and mutant variants

Studies with other bacterial ATP synthases have revealed that the β subunit typically shows higher affinity for ATP than ADP, with dissociation constants in the micromolar range. The binding energetics are significantly influenced by magnesium concentration and pH, reflecting the physiological regulation of ATP synthase activity .

How can researchers address solubility issues with recombinant F. johnsoniae ATP synthase β subunit?

Solubility challenges are common when working with recombinant ATP synthase subunits. The following strategies have proven effective for improving the solubility of F. johnsoniae β subunit:

• Expression optimization:

  • Reduce induction temperature to 16-18°C

  • Decrease inducer concentration (0.1-0.2 mM IPTG)

  • Use auto-induction media for gradual protein expression

• Buffer optimization:

  • Screen various pH conditions (pH 6.5-8.5)

  • Test different salt concentrations (100-500 mM NaCl)

  • Include solubility enhancers: 5-10% glycerol, 0.1% non-ionic detergents, or 50-100 mM arginine

• Fusion partner approach:

  • MBP (maltose-binding protein) fusion typically increases solubility significantly

  • SUMO or thioredoxin fusions can also improve folding and solubility

  • Include a cleavable linker for removal of the fusion partner after purification

• Co-expression strategies:

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

  • Co-express with other ATP synthase subunits that naturally interact with β

These approaches have shown success rates of 60-80% in improving the solubility of ATP synthase subunits from various bacterial species .

What strategies address low yield in recombinant β subunit production?

Low yield of recombinant F. johnsoniae ATP synthase β subunit can be addressed through systematic optimization:

• Strain selection:

  • Compare BL21(DE3) with derivatives like Rosetta (for rare codons) or C41/C43 (for toxic proteins)

  • Consider Lemo21(DE3) for tunable expression level

• Growth media optimization:

  • Rich media (TB, 2xYT) typically increase yields 2-3 fold over standard LB

  • Supplementation with trace elements and vitamin mix can further improve yields

  • Fed-batch strategies maintain optimal growth conditions

• Harvest timing optimization:

  • Extended expression time (24-48 hours) at lower temperatures often improves yield

  • Monitor protein accumulation by small-scale sampling and SDS-PAGE analysis

• Cell lysis improvement:

  • Optimize lysis buffer composition (detergents, salt concentration)

  • Consider enzymatic pre-treatment (lysozyme) before mechanical disruption

  • Multiple gentle lysis cycles may increase recovery of soluble protein

• Purification recovery enhancement:

  • Optimize binding and elution conditions for affinity chromatography

  • Include ATP (1-2 mM) in buffers to stabilize the protein during purification

  • Consider on-column refolding for proteins recovered from inclusion bodies

Systematic application of these strategies has been shown to increase yields of recombinant ATP synthase subunits from sub-milligram to 5-10 mg per liter of culture .

How can researchers verify species-specific characteristics versus expression artifacts?

Distinguishing genuine species-specific characteristics of F. johnsoniae ATP synthase β subunit from artifacts related to recombinant expression requires rigorous controls and comparative analyses:

• Multi-system expression comparison:

  • Express the same construct in different systems (E. coli, yeast, cell-free)

  • Compare biochemical properties to identify system-independent characteristics

• Homolog comparison:

  • Express β subunits from related and distant bacterial species under identical conditions

  • Characteristics conserved across phylogenetically related species are likely genuine

• Structural verification:

  • Compare secondary structure content using circular dichroism

  • Verify tertiary structure integrity through intrinsic fluorescence spectroscopy

  • Conduct thermal denaturation studies to compare stability profiles

• Functional benchmarking:

  • Compare nucleotide binding affinities with published data for related species

  • Assess ATPase activity under standardized conditions

  • Evaluate reconstitution efficiency with other ATP synthase subunits

• Native protein comparison (when possible):

  • Isolate native ATP synthase from F. johnsoniae

  • Compare properties of the native β subunit with the recombinant version

These approaches create a matrix of data points that collectively distinguish genuine species-specific characteristics from expression-related artifacts .

How can the recombinant β subunit contribute to drug development targeting bacterial ATP synthases?

The recombinant F. johnsoniae ATP synthase β subunit serves as a valuable tool for anti-bacterial drug discovery, particularly given the emerging importance of ATP synthase as a drug target against bacteria like Mycobacterium tuberculosis :

• High-throughput screening platforms:

  • Develop fluorescence-based assays using the recombinant β subunit to screen compound libraries

  • Establish thermal shift assays to identify compounds that bind and stabilize specific conformations

  • Create competitive binding assays to identify molecules that displace nucleotides

• Structure-based drug design:

  • Use crystal structures of the recombinant protein for in silico screening

  • Identify species-specific pockets that differ from human ATP synthase for selective targeting

  • Design compounds that interfere with β subunit interactions with other ATP synthase components

• Resistance mechanism studies:

  • Generate mutations associated with drug resistance in the recombinant protein

  • Study how these mutations affect drug binding without requiring the pathogenic organism

  • Develop combination approaches to overcome resistance mechanisms

• Comparative species analysis:

  • Study β subunits from multiple bacterial species to develop broad-spectrum inhibitors

  • Identify conserved features for pan-bacterial targeting

This research direction has significant potential given that ATP synthase inhibitors like bedaquiline have already proven successful against drug-resistant tuberculosis, suggesting similar approaches could work for other bacterial pathogens .

What novel biotechnological applications could utilize engineered versions of the recombinant β subunit?

Engineered variants of the recombinant F. johnsoniae ATP synthase β subunit offer exciting biotechnological opportunities:

• Biosensors for ATP/ADP ratios:

  • Engineer the β subunit with fluorescent proteins or dyes that respond to nucleotide binding

  • Develop real-time cellular energy state monitors

  • Create diagnostic tools for metabolic disorders

• Nanomotor development:

  • Utilize the natural rotary mechanism of ATP synthase for nanoscale mechanical devices

  • Engineer the β subunit to respond to alternative energy sources

  • Create hybrid biological-synthetic nanomachines

• Bioenergetic system enhancements:

  • Engineer the β subunit for improved catalytic efficiency

  • Develop variants with altered ion specificity or coupling efficiency

  • Create systems for enhanced biological energy conversion

• Protein interaction scaffolds:

  • Utilize the stable structure of the β subunit as a scaffold for presenting functional domains

  • Develop protein-based materials with controlled assembly properties

  • Create multi-enzyme complexes with improved catalytic efficiency

These applications leverage the natural properties of the ATP synthase β subunit while extending functionality through protein engineering approaches .

How might systems biology approaches incorporate recombinant β subunit research to understand cellular energetics?

Systems biology approaches integrating recombinant F. johnsoniae ATP synthase β subunit research can provide comprehensive understanding of cellular energetics:

• Multi-omics integration:

  • Correlate proteomic changes in energy metabolism with ATP synthase modifications

  • Integrate transcriptomic data to understand regulation of energy production

  • Map metabolomic shifts resulting from altered ATP synthase activity

• Mathematical modeling of energy dynamics:

  • Develop kinetic models incorporating parameters derived from recombinant protein studies

  • Create predictive models of cellular responses to energy limitation

  • Simulate evolutionary adaptations in energy metabolism

• Synthetic biology applications:

  • Design minimal cells with optimized energy production systems

  • Engineer microorganisms with enhanced ATP production for biotechnological applications

  • Create synthetic regulatory circuits controlling energy metabolism

• Comparative physiology and adaptation studies:

  • Understand how ATP synthase variations contribute to ecological adaptation

  • Study energy metabolism across extreme environments

  • Investigate evolutionary conservation and divergence of bioenergetic systems

This systems-level understanding could provide insights into fundamental aspects of life's energetic requirements and offer new approaches for engineering microorganisms for biotechnological applications .

Frequently Asked Questions for Researchers: Recombinant Flavobacterium johnsoniae ATP Synthase Subunit Beta (atpD), Partial

This comprehensive collection addresses key research questions about the recombinant F. johnsoniae ATP synthase β subunit, providing methodological insights and experimental guidance based on scientific literature and comparable research on ATP synthases. The information is organized from fundamental concepts to advanced applications to facilitate research at various expertise levels.

What is the structure and function of ATP synthase β subunit (atpD) in F. johnsoniae?

The ATP synthase β subunit (atpD) is a critical catalytic component of the F1 sector in F-type ATP synthases. Similar to its counterpart in other bacterial species, the F. johnsoniae β subunit forms part of the α3β3 catalytic hexamer within the F1 region. This subunit contains nucleotide-binding domains essential for ATP synthesis and hydrolysis.

The β subunit works cooperatively with the α subunit to create three catalytic sites at their interfaces where ATP synthesis occurs. Unlike the α subunit which plays primarily a structural role, the β subunit directly participates in catalysis through conformational changes driven by the rotation of the central γ-stalk . This rotation is coupled to proton translocation through the membrane-embedded FO portion, creating the mechanical energy needed for ATP synthesis.

The F. johnsoniae β subunit shares the characteristic Walker A and B motifs found in other ATP synthases, which are critical for nucleotide binding and hydrolysis. The catalytic mechanism involves a series of conformational changes that correspond to different affinity states for nucleotides (empty, loose, and tight binding states), ultimately resulting in ATP synthesis or hydrolysis .

How does the β subunit (atpD) differ structurally and functionally from the α subunit (atpA)?

While the α and β subunits share considerable sequence homology and structural similarity, they serve distinct roles in ATP synthase function:

• Catalytic activity: The β subunit contains the primary catalytic sites for ATP synthesis/hydrolysis, while the α subunit plays a more regulatory role in nucleotide binding but lacks catalytic activity .

• Nucleotide binding characteristics: Both subunits bind nucleotides, but the β subunit exhibits catalytic turnover, whereas the α subunit's nucleotide binding sites are typically non-catalytic and may serve regulatory functions .

• Sequence features: The β subunit contains specific conserved motifs including the DELSEED sequence that interacts with the γ subunit during rotational catalysis, which is absent in the α subunit .

• Structural dynamics: During catalysis, the β subunit undergoes more substantial conformational changes than the α subunit, reflecting its direct involvement in the energy conversion process .

These differences influence how recombinant versions of each subunit behave in experimental settings, with the β subunit typically demonstrating measurable ATPase activity when correctly folded, while the α subunit does not .

What approaches can verify the structural integrity of a recombinant partial β subunit?

Ensuring the structural integrity of a recombinant partial β subunit is crucial for reliable experimental outcomes. Several complementary approaches can be employed:

• SDS-PAGE analysis: Confirms the expected molecular weight and initial purity (typically >85% for commercial preparations) .

• Circular dichroism (CD) spectroscopy: Verifies secondary structure elements match theoretical predictions for the β subunit.

• Limited proteolysis: Partial β subunits with correct folding demonstrate characteristic proteolytic resistance patterns.

• Intrinsic fluorescence spectroscopy: Monitors the environment of tryptophan residues to confirm tertiary structure integrity.

• Thermal shift assays: Properly folded partial β subunits show cooperative unfolding with characteristic transition temperatures.

• Nucleotide binding assays: Using fluorescent ATP analogs or isothermal titration calorimetry to confirm functional binding capacity.

• Activity assays: While partial constructs may not show full activity, they should retain some degree of nucleotide interaction capability that can be measured through ATPase activity assays .

For partial recombinant constructs, it's especially important to verify that the produced fragment contains complete functional domains rather than truncated ones, which may lead to misfolded structures with compromised stability .

What expression systems and conditions yield optimal results for recombinant F. johnsoniae ATP synthase β subunit?

Based on comparable research with ATP synthase subunits, the following expression systems and conditions yield optimal results:

For E. coli expression systems, which have been successfully used for ATP synthase α subunits from F. johnsoniae, the following optimizations are recommended :

• Use rich media (Terrific Broth or 2xYT) supplemented with 1% glucose and 5% glycerol

• Include 5-10 mM MgSO4 in the media to support proper folding

• Add 0.5-1.0 mM ATP to lysis buffers to stabilize nucleotide-binding regions

• Co-express with molecular chaperones (GroEL/GroES) to improve folding efficiency

Expression in E. coli systems typically results in higher yields, with proper protein folding verified through activity assays that specifically target the catalytic function rather than just structural integrity .

What purification protocol provides the highest purity while maintaining the functionality of the β subunit?

A multi-step purification protocol optimized for ATP synthase β subunit ensures both high purity and preserved functionality:

• Initial cell lysis and clarification:

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM ATP, 5 mM MgCl2, protease inhibitor cocktail

  • Sonication or high-pressure homogenization (15,000-20,000 psi, 3 passes)

  • Centrifugation at 20,000 × g for 30 min, followed by 100,000 × g for 1 hour

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) using His-tagged constructs

  • Gradual imidazole gradient (20-250 mM) to minimize co-purification of contaminants

  • Include 0.5 mM ATP and 2 mM MgCl2 in buffers to stabilize the protein

• Ion exchange chromatography:

  • Q-Sepharose column with 50-500 mM NaCl gradient

  • Buffer containing 20 mM Tris-HCl pH a8.0, 10% glycerol, 0.5 mM ATP, 2 mM MgCl2, 1 mM DTT

• Size exclusion chromatography:

  • Superdex 200 column equilibrated with 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 0.5 mM ATP, 2 mM MgCl2, 1 mM DTT

This protocol consistently yields protein with >85% purity as verified by SDS-PAGE, comparable to the purity levels reported for commercial recombinant α subunit preparations . The inclusion of ATP and magnesium throughout the purification process is critical for maintaining the native conformation and stability of the β subunit .

How can researchers distinguish between full-length and partial recombinant β subunit in terms of functional characteristics?

Distinguishing between full-length and partial recombinant β subunit requires careful characterization of both structural and functional properties:

• Size verification:

  • SDS-PAGE analysis to confirm molecular weight differences

  • Mass spectrometry to determine precise molecular masses and sequence coverage

• Domain-specific functional assays:

  • Nucleotide binding assays to compare affinity constants (KD)

  • ATP hydrolysis activity measurements (partial constructs typically show reduced but detectable activity)

  • Binding studies with partner subunits from the ATP synthase complex

• Structural stability comparison:

  • Thermal denaturation profiles (partial constructs often show lower melting temperatures)

  • Chemical denaturation using urea or guanidinium hydrochloride

  • Limited proteolysis patterns (partial constructs may show different digestion patterns)

• Reconstitution efficiency:

  • Ability to form higher-order assemblies with complementary subunits

  • Analysis by native PAGE or analytical ultracentrifugation

Research has shown that partial β subunit constructs that contain complete functional domains can retain specific activities, particularly nucleotide binding, even when they lack the full complement of intersubunit interaction regions . For experimental planning, it's critical to understand which specific functional characteristics are preserved in the partial construct being used.

What storage conditions maximize the stability and shelf-life of the recombinant β subunit?

Optimal storage conditions for recombinant F. johnsoniae ATP synthase β subunit parallel those established for the α subunit, with some modifications to account for the β subunit's catalytic nature:

• Short-term storage (1-2 weeks):

  • Store at 4°C in buffer containing 20 mM Tris-HCl pH 7.5-8.0, 100-150 mM NaCl, 10% glycerol, 1 mM DTT, 0.5 mM ATP, and 2 mM MgCl2

  • Addition of 0.02% sodium azide prevents microbial growth

• Medium-term storage (1-6 months):

  • Store at -20°C with 20-50% glycerol as a cryoprotectant

  • Aliquot in small volumes to avoid repeated freeze-thaw cycles

• Long-term storage (>6 months):

  • Store at -80°C as lyophilized powder or in solution with 50% glycerol

  • For lyophilized storage, include 5% trehalose or sucrose as a lyoprotectant

The shelf life of recombinant proteins is significantly influenced by storage conditions:

As noted in product information for ATP synthase subunits, repeated freezing and thawing significantly reduces protein stability and should be avoided . The inclusion of nucleotides (ATP) in storage buffers has been shown to enhance stability by promoting native conformation through nucleotide binding .

What reconstitution protocols are recommended for lyophilized recombinant β subunit?

For optimal reconstitution of lyophilized recombinant F. johnsoniae ATP synthase β subunit:

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

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

• Allow the protein to rehydrate completely at room temperature for 10-15 minutes with occasional gentle mixing

• Add glycerol to a final concentration of 20-50% for storage stability if preparing for long-term storage

• For functional studies, consider adding:

  • 2-5 mM MgCl2

  • 0.5-1 mM ATP

  • 20 mM Tris-HCl pH 8.0

  • 100 mM NaCl

The addition of nucleotides and magnesium ions during reconstitution has been shown to enhance stability by promoting native conformation. Research has demonstrated that nucleotide binding is crucial for proper folding and stability of ATP synthase subunits, while ATP hydrolysis appears less critical for structural integrity .

After reconstitution, centrifuge briefly at 10,000 × g for 1 minute to remove any insoluble material. Prepare working aliquots in volumes appropriate for single experiments to avoid repeated freeze-thaw cycles, which significantly reduce protein stability and activity.

How does the partial nature of the recombinant β subunit affect its stability compared to the full-length protein?

The partial nature of recombinant F. johnsoniae ATP synthase β subunit has significant implications for stability compared to the full-length protein:

• Exposed hydrophobic surfaces: Partial constructs may expose hydrophobic regions that would normally be buried in interfaces with other subunits or domains, leading to reduced solubility and increased aggregation propensity.

• Altered nucleotide binding characteristics: If the partial construct includes complete nucleotide-binding domains, stability can be enhanced by adding nucleotides to buffers. Studies have shown that nucleotide binding plays a crucial role in stabilizing ATP synthase subunits .

• Reduced thermal stability: Partial constructs typically exhibit lower melting temperatures (Tm) in thermal denaturation studies, often by 5-10°C compared to full-length proteins.

• Modified salt sensitivity: Partial constructs may show altered sensitivity to ionic strength due to changed surface charge distribution.

To mitigate these stability challenges, several approaches are recommended:

• Include stabilizing additives such as glycerol (10-20%), arginine (50-100 mM), or non-detergent sulfobetaines

• Maintain reducing conditions with 1-5 mM DTT or 2-10 mM β-mercaptoethanol

• Add nucleotides (0.5-1 mM ATP) and divalent cations (2-5 mM MgCl2) to stabilize nucleotide-binding domains

• Store at higher protein concentrations (>1 mg/mL) to reduce surface-dependent denaturation

How can the recombinant β subunit be used to study ATP synthase assembly pathways?

The recombinant F. johnsoniae ATP synthase β subunit serves as a valuable tool for investigating assembly pathways of the complete ATP synthase complex:

• In vitro reconstitution studies:

  • Mix purified recombinant β subunit with other purified subunits in controlled ratios

  • Monitor assembly intermediates using native PAGE, analytical ultracentrifugation, or size exclusion chromatography

  • Investigate the role of nucleotides in promoting assembly by varying nucleotide concentrations and types

• Assembly kinetics analysis:

  • Use time-resolved techniques such as stopped-flow fluorescence or FRET to monitor assembly rates

  • Investigate temperature, pH, and ionic strength effects on assembly pathways

  • Apply mathematical modeling to determine rate-limiting steps in assembly

• Mutational analysis of assembly determinants:

  • Introduce specific mutations to interface regions and monitor effects on assembly efficiency

  • Identify critical residues required for proper subunit-subunit interactions

  • Create chimeric constructs to determine species-specific assembly requirements

Recent research using laser induced liquid bead ion desorption (LILBID) mass spectrometry has demonstrated that nucleotide binding is crucial for in vitro F1 assembly, while ATP hydrolysis appears less critical for the assembly process . This approach can be extended to F. johnsoniae ATP synthase to identify species-specific assembly characteristics and compare them with model organisms.

What functional assays can verify the catalytic activity of the recombinant β subunit?

Several complementary assays can verify the catalytic activity of recombinant F. johnsoniae ATP synthase β subunit:

• ATP hydrolysis assays:

  • Colorimetric phosphate detection: Using malachite green or molybdate-based assays to measure released inorganic phosphate

  • Coupled enzyme assays: Using pyruvate kinase and lactate dehydrogenase to couple ATP hydrolysis to NADH oxidation, monitored at 340 nm

  • Luciferase-based ATP consumption: Measuring remaining ATP after incubation with the recombinant protein

• Nucleotide binding assays:

  • Fluorescence-based binding: Using fluorescent ATP analogs like TNP-ATP or MANT-ATP

  • Isothermal titration calorimetry (ITC): Providing comprehensive thermodynamic binding parameters

  • Surface plasmon resonance (SPR): Offering real-time binding kinetics

• Partial reaction characterization:

  • Pi exchange assays: Measuring ATP synthase-catalyzed exchange between medium Pi and ATP

  • Tryptophan fluorescence changes: Monitoring conformational changes associated with catalytic steps

For partial recombinant β subunit constructs, activity is typically lower than full-length protein and may require optimization of assay sensitivity. Important considerations include:

• Including Mg2+ (typically 5 mM) as a cofactor for ATP hydrolysis

• Testing activity across a pH range (7.0-8.5) to identify optimal conditions

• Evaluating the effects of various ATP concentrations to determine kinetic parameters (KM, Vmax)

• Including appropriate controls such as heat-inactivated protein and reactions with specific inhibitors

How can the recombinant β subunit be used to screen for inhibitors targeting bacterial ATP synthases?

The recombinant F. johnsoniae ATP synthase β subunit provides an excellent platform for screening potential inhibitors targeting bacterial ATP synthases:

• High-throughput screening approaches:

  • ATP hydrolysis inhibition assays: Measuring reduced phosphate release in the presence of potential inhibitors

  • Thermal shift assays: Detecting changes in protein thermal stability upon inhibitor binding

  • Fluorescence-based nucleotide displacement: Monitoring displacement of fluorescent nucleotide analogs by inhibitor compounds

• Structure-based screening:

  • Molecular docking: Using homology models or crystal structures to predict binding of virtual compound libraries

  • Fragment-based screening: Identifying small molecular fragments that bind to specific sites on the β subunit

  • NMR-based screening: Detecting binding events through chemical shift perturbations

• Selectivity profiling:

  • Parallel screening: Testing compounds against bacterial and human/mammalian ATP synthase components

  • Differential scanning fluorimetry: Comparing stabilization effects across species

  • Comparative inhibition kinetics: Determining IC50 values across diverse bacterial species

• Validation and mechanism studies:

  • Site-directed mutagenesis: Confirming binding sites by mutating predicted contact residues

  • Crystallography or cryo-EM: Resolving structures of inhibitor-bound complexes

  • Molecular dynamics simulations: Exploring dynamic aspects of inhibitor interactions

The importance of ATP synthase as a drug target has been highlighted by the success of bedaquiline against Mycobacterium tuberculosis . Similar approaches using recombinant F. johnsoniae β subunit could lead to the development of novel antibiotics targeting this essential enzyme in other bacterial pathogens.

How do mutations in the catalytic sites of the β subunit affect nucleotide binding and hydrolysis?

Site-directed mutagenesis of the recombinant F. johnsoniae ATP synthase β subunit provides critical insights into structure-function relationships:

• Key catalytic residues and their effects when mutated:

• Biochemical characterization techniques:

  • Steady-state kinetics to determine KM and kcat changes

  • Pre-steady-state kinetics to identify rate-limiting steps

  • Nucleotide binding studies using ITC or fluorescence approaches

  • Thermal stability assays to assess structural impacts of mutations

• Mechanistic insights:

  • Mutations in the Walker A motif primarily affect nucleotide binding affinity

  • Walker B mutations typically permit binding but impair catalysis

  • DELSEED motif mutations affect the coupling between catalysis and mechanical rotation

  • Interface residues impact communication between adjacent subunits

These mutational studies reveal that the catalytic mechanism involves precise coordination between nucleotide binding, conformational changes, and inter-subunit communication. The β subunit's catalytic properties can be fine-tuned through specific amino acid substitutions, providing insights into evolutionary adaptations and potential drug targets .

How does the recombinant partial β subunit interact with other ATP synthase components in reconstitution experiments?

Reconstitution experiments using the recombinant F. johnsoniae ATP synthase β subunit reveal critical insights into subunit-subunit interactions:

• Interaction with α subunit:

  • Forms stable α3β3 hexamers when combined in equimolar ratios

  • Requires the presence of nucleotides (ATP or non-hydrolyzable analogs) for efficient assembly

  • Assembly can be monitored by native PAGE, analytical ultracentrifugation, or FRET between labeled subunits

• Interaction with γ subunit:

  • The central cavity of α3β3 hexamer accommodates the γ subunit

  • DELSEED motif in β subunit forms critical contacts with γ during rotation

  • Partial β constructs may show reduced interaction capability depending on which domains are present

• Interaction with minor subunits (δ, ε):

  • Primarily indirect interactions mediated through α and γ subunits

  • Complete F1 assembly follows a sequential pathway with defined intermediates

• Methodological approaches:

  • Sequential addition of subunits with monitoring by native PAGE or gel filtration

  • Single-molecule studies using fluorescently labeled components

  • Cryo-EM analysis of reconstituted complexes at various assembly stages

Research has shown that nucleotide binding plays a critical role in promoting proper assembly, while ATP hydrolysis is less critical for the assembly process itself . This suggests that non-hydrolyzable ATP analogs can be used during reconstitution to stabilize intermediates without promoting premature complex disassembly.

For partial β subunit constructs, reconstitution efficiency depends heavily on which domains are present and whether key interaction surfaces are intact. Compensatory strategies, such as using linker peptides or fusion proteins, can sometimes overcome limitations of partial constructs .

How can evolutionary analysis of the β subunit across bacterial species inform structure-function relationships?

Comparative evolutionary analysis of ATP synthase β subunits across bacterial species provides valuable insights into structure-function relationships:

• Sequence conservation patterns:

  • Catalytic residues show near-absolute conservation across all bacterial species

  • Interface residues interacting with other subunits show lineage-specific conservation patterns

  • Variable regions often correlate with species-specific regulatory mechanisms or environmental adaptations

• Structural adaptations:

  • Thermophilic bacteria show increased ionic interactions and disulfide bonds in β subunits

  • Acidophiles display modifications in surface charge distribution

  • Alkaliphiles exhibit adaptations in proton-conducting pathways

• Co-evolution analysis:

  • Correlated mutations between β subunit and other ATP synthase components reveal functional coupling

  • Identification of evolutionarily coupled residue networks provides insight into allosteric communication

  • Statistical coupling analysis can predict functionally important but experimentally unverified residues

• Methodological approaches:

  • Multiple sequence alignment of β subunits from diverse bacterial species

  • Phylogenetic analysis to trace evolutionary trajectories

  • Structural mapping of conservation patterns

  • Molecular dynamics simulations of species-specific variants

Applying these evolutionary insights to F. johnsoniae ATP synthase β subunit can reveal unique adaptations potentially related to the organism's lifestyle and ecological niche. For instance, comparative analysis between F. johnsoniae and other bacterial species like Mycobacterium has revealed differences in nucleotide binding affinities and catalytic properties that may reflect different energetic requirements .

This evolutionary perspective also informs drug development by identifying species-specific features that can be targeted while avoiding interference with human ATP synthase, thereby reducing potential side effects .

What strategies can address protein aggregation issues with recombinant β subunit?

Addressing protein aggregation issues with recombinant F. johnsoniae ATP synthase β subunit requires a systematic approach:

• Expression optimization:

  • Reduce expression temperature (16-18°C) to slow protein synthesis and improve folding

  • Lower inducer concentration (0.1-0.2 mM IPTG) to prevent overwhelming cellular folding machinery

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ) to assist proper folding

• Buffer optimization:

  • Screen pH range (typically pH 6.5-8.5) to find conditions minimizing aggregation

  • Test various salt concentrations (100-500 mM NaCl) to optimize electrostatic interactions

  • Include solubility enhancers:

    • 5-20% glycerol to stabilize hydrophobic interactions

    • 50-100 mM arginine or glutamate to prevent aggregation

    • 0.5-1 mM ATP and 2-5 mM MgCl2 to stabilize nucleotide-binding regions

• Additive screening:

  • Non-ionic detergents (0.01-0.05% Triton X-100 or NP-40)

  • Non-detergent sulfobetaines (NDSB-201, NDSB-256)

  • Amino acid additives (proline, histidine)

  • Osmolytes (trehalose, sucrose, betaine)

• Protein engineering approaches:

  • Fusion with solubility-enhancing partners (MBP, SUMO, thioredoxin)

  • Surface entropy reduction through mutation of flexible charged residues

  • Truncation of highly aggregation-prone regions while preserving functional domains

For partial constructs, particular attention should be paid to ensuring that domain boundaries are appropriately designed to maintain structural integrity and avoid exposing hydrophobic regions that would normally be buried .

How can researchers increase the yield and purity of recombinant β subunit?

Optimizing yield and purity of recombinant F. johnsoniae ATP synthase β subunit requires attention to multiple experimental factors:

• Strain and vector optimization:

  • Compare BL21(DE3) with derivatives like Rosetta (for rare codons) or C41/C43 (for membrane proteins)

  • Optimize codon usage for F. johnsoniae sequences in expression host

  • Test different promoter strengths (T7, tac, ara) for optimal expression level

  • Include appropriate fusion tags (His6, GST, MBP) with efficient cleavage sites

• Culture optimization:

  • Use rich media (TB, 2xYT) with glucose supplementation to prevent leaky expression

  • Implement fed-batch strategies to maintain optimal growth conditions

  • Optimize cell density at induction (typically OD600 = 0.6-0.8)

  • Include specific additives in growth media (5-10 mM MgSO4, trace metals)

• Purification refinement:

  • Optimize lysis conditions (buffer composition, mechanical method, enzymatic pre-treatment)

  • Implement two-phase extraction systems to remove contaminants early

  • Use orthogonal chromatography techniques:

    • Affinity chromatography with optimized binding/elution conditions

    • Ion exchange chromatography with shallow gradients for better separation

    • Hydrophobic interaction chromatography as an alternative purification step

    • Size exclusion chromatography as a final polishing step

• Workflow optimization:

  • Minimize time between cell harvest and initial purification steps

  • Include stabilizing agents throughout purification (nucleotides, glycerol)

  • Optimize protein concentration methods to prevent aggregation

  • Consider on-column refolding for proteins recovered from inclusion bodies

These optimizations can increase yields from sub-milligram to 5-10 mg per liter of culture while achieving >90% purity, comparable to commercial preparations of ATP synthase subunits .

How can researchers troubleshoot inconsistent activity results with recombinant β subunit?

Inconsistent activity results with recombinant F. johnsoniae ATP synthase β subunit can be methodically addressed:

• Protein quality assessment:

  • Verify protein integrity by SDS-PAGE and mass spectrometry

  • Check for degradation products or truncated forms

  • Assess aggregation state by dynamic light scattering or size exclusion chromatography

  • Confirm proper folding using circular dichroism or fluorescence spectroscopy

• Assay optimization:

  • Carefully control buffer conditions (pH, ionic strength)

  • Ensure consistent cofactor concentrations (Mg2+, ATP)

  • Validate assay reagents quality and prepare fresh working solutions

  • Establish standard curves with each experiment

  • Include positive controls (commercial ATP hydrolysis standards)

• Environmental variables:

  • Control temperature precisely during assays (typically 25°C or 37°C)

  • Protect samples from light when using photosensitive detection methods

  • Minimize freeze-thaw cycles by using fresh aliquots

  • Control reaction times precisely with consistent stopping methods

• Activity reconciliation approaches:

  • Characterize the kinetic parameters systematically (KM, Vmax, optimal pH)

  • Compare multiple activity assay methods (colorimetric, coupled enzyme, direct detection)

  • Normalize activity to active site concentration rather than total protein

  • Consider the influence of the partial nature of the construct on activity measurements

For partial β subunit constructs, activity is often lower and more variable than full-length proteins. Establishing a standard operating procedure with rigorous quality controls at each step helps ensure reproducible results across experiments .

How might the recombinant β subunit contribute to the development of novel antibiotics?

The recombinant F. johnsoniae ATP synthase β subunit offers significant potential for antibiotic development:

• Target validation approaches:

  • Structure-based drug design targeting unique features of bacterial β subunits

  • Identification of allosteric sites that disrupt catalysis or assembly

  • Development of high-throughput screening assays using recombinant protein

  • Testing of combination approaches that synergize with existing antibiotics

• Inhibition mechanism exploration:

  • Competitive inhibitors targeting the ATP binding site

  • Allosteric inhibitors disrupting conformational changes

  • Interface inhibitors preventing proper assembly with other subunits

  • Covalent modifiers targeting exposed cysteine residues specific to bacterial enzymes

• Species-specific targeting:

  • Comparative analysis across bacterial pathogens to identify conserved targets

  • Exploitation of structural differences between bacterial and human ATP synthases

  • Development of narrow-spectrum antibiotics targeting specific bacterial clades

• Resistance mechanism studies:

  • Identifying potential resistance mutations using recombinant protein models

  • Exploring evolutionary constraints on resistance development

  • Designing inhibitor combinations to minimize resistance emergence

The success of bedaquiline as an ATP synthase inhibitor against drug-resistant Mycobacterium tuberculosis demonstrates the clinical potential of this approach . F. johnsoniae studies could reveal binding sites or mechanisms applicable to a broader range of bacterial pathogens, potentially addressing the growing challenge of antimicrobial resistance.

How can systems biology approaches integrate recombinant β subunit research into broader understanding of cellular energetics?

Systems biology approaches integrating recombinant F. johnsoniae ATP synthase β subunit research provide comprehensive understanding of cellular energetics:

• Multi-omics integration:

  • Correlate proteomic changes in energy metabolism with ATP synthase modifications

  • Integrate transcriptomic data to understand regulation of ATP synthase expression

  • Map metabolomic shifts resulting from altered ATP synthase activity

  • Develop genome-scale metabolic models incorporating experimentally determined enzyme parameters

• Mathematical modeling of energy dynamics:

  • Create kinetic models of ATP synthesis and consumption

  • Simulate the effects of environmental perturbations on energy homeostasis

  • Predict cellular responses to energy limitation based on experimental data

  • Model evolutionary adaptations in energy metabolism across bacterial species

• Network analysis:

  • Identify interaction networks centered on ATP synthase

  • Map regulatory networks controlling energy production

  • Analyze flux distributions under various energy states

  • Study emergent properties of integrated energy production systems

• Synthetic biology applications:

  • Design minimal cells with optimized energy production systems

  • Engineer microorganisms with enhanced ATP production for biotechnological applications

  • Create synthetic regulatory circuits controlling energy metabolism

These integrative approaches could reveal how F. johnsoniae's energy metabolism contributes to its distinctive biological properties, such as gliding motility, and how ATP synthase has evolutionarily adapted to support these functions .

What novel biotechnological applications could utilize engineered versions of the recombinant β subunit?

Engineered versions of the recombinant F. johnsoniae ATP synthase β subunit offer exciting biotechnological opportunities:

• Biosensor development:

  • Engineer the β subunit with fluorescent proteins or dyes that respond to nucleotide binding

  • Develop real-time cellular energy state monitors

  • Create diagnostic tools for detecting ATP/ADP ratio changes in disease states

  • Design environmental biosensors for detecting specific compounds that affect ATP synthesis

• Nanomotor applications:

  • Utilize the natural rotary mechanism for nanoscale mechanical devices

  • Create hybrid biological-synthetic nanomachines powered by ATP

  • Develop molecular motors with enhanced efficiency or altered specificity

  • Engineer responsive nanomachines that change behavior based on environmental signals

• Bioenergetic system enhancements:

  • Engineer the β subunit for improved catalytic efficiency

  • Develop variants with altered ion specificity or coupling efficiency

  • Create systems for enhanced biological energy conversion in biotechnology applications

  • Design ATP synthases with modified regulatory properties for industrial processes

• Biopharmaceutical applications:

  • Develop ATP synthase-based drug delivery systems

  • Create protein scaffolds for presenting therapeutic epitopes

  • Design β subunit variants as adjuvants for vaccine development

  • Engineer self-assembling nanostructures for medical applications

These applications leverage the natural properties of the ATP synthase β subunit while extending functionality through protein engineering approaches, potentially leading to transformative technologies at the interface of biology and nanotechnology .