Recombinant Koribacter versatilis ATP synthase subunit b (atpF)

What are the structural characteristics of Koribacter versatilis ATP synthase subunit b compared to other bacterial species?

The Koribacter versatilis ATP synthase subunit b exhibits structural features that are both conserved across bacterial species and unique to acidophilic bacteria:

Research on bacterial ATP synthases shows that while the peripheral stalk in Koribacter versatilis is structurally simpler than in eukaryotic mitochondria, it maintains the functional architecture necessary for ATP synthesis in acidic environments . Unlike the mitochondrial structure where the peripheral stalk provides significant rigidity, the bacterial peripheral stalk (including atpF) displays more flexibility, which may be important for adaptation to environmental stresses .

What expression systems are most effective for producing recombinant Koribacter versatilis atpF?

E. coli expression systems are predominantly used for recombinant production of Koribacter versatilis atpF due to their efficiency and scalability. Several specific approaches have been documented:

For optimal results with membrane proteins like atpF:

• Use E. coli strains specifically designed for membrane protein expression (C41/C43)

• Consider fusion partners that enhance solubility

• Implement controlled induction protocols to prevent toxicity

Research indicates that E. coli has been successfully used to express related ATP synthase components from Koribacter versatilis, with protocols similar to those used for the ATP synthase subunit a (atpB) . Temperature optimization during induction has been shown to significantly impact the solubility of membrane-associated proteins .

What are the optimal buffer conditions and storage parameters for maintaining the stability of purified recombinant Koribacter versatilis atpF?

The stability of purified recombinant Koribacter versatilis atpF is highly dependent on appropriate buffer conditions and storage parameters:

Recommended buffer compositions:

Storage recommendations:

• Store at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

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

• Repeated freezing and thawing is not recommended

Research with similar ATP synthase components demonstrates that proper buffer selection significantly impacts both structural integrity and functional activity. The addition of glycerol and trehalose as cryoprotectants has been shown to maintain protein stability during freeze-thaw cycles .

How can researchers address contradictory data when studying ATP synthase activities in Koribacter versatilis?

When encountering contradictory data in Koribacter versatilis ATP synthase research, a systematic approach is necessary:

Methodological framework for resolving contradictions:

• Verify experimental conditions:

  • Ensure comparable protein concentrations across experiments

  • Standardize buffer compositions and pH conditions

  • Control temperature parameters precisely

• Implement validation strategies:

  • Use multiple complementary techniques (e.g., biochemical assays, structural studies)

  • Employ different detection methods for the same parameter

  • Analyze both in vitro and in vivo activities when possible

• Apply targeted statistical approaches:

  • Perform paired statistical tests for direct comparisons

  • Use linear discriminant analysis for complex datasets

  • Consider Bayesian approaches for integrating prior knowledge

• Consider context-dependent factors:
  Research shows that seemingly contradictory results may arise from:

  • Different oligomeric states of the protein

  • Varying lipid environments affecting membrane protein function

  • Post-translational modifications altering activity profiles

As noted in literature on contradictory data analysis: "Contradictions as a data quality indicator are typically understood as impossible combinations of values in interdependent data items. While the handling of a single dependency between two data items is well established, for more complex interdependencies, there is not yet a common notation or structured evaluation method established" . This highlights the importance of employing multiple analytical approaches when confronted with contradictory ATP synthase activity data.

What techniques are most effective for analyzing interactions between atpF and other ATP synthase subunits in Koribacter versatilis?

Several advanced techniques have proven effective for investigating the interactions between atpF and other ATP synthase subunits:

Research demonstrates that bacterial ATP synthase complexes can be effectively studied using complementary approaches. For example, cryo-EM has successfully revealed the structural organization of bacterial ATP synthases, showing that "the peripheral stalk is structurally simpler and more flexible than in yeast mitochondria" .

For accurate analysis of atpF interactions, it's crucial to maintain native-like conditions during experiments, as the membrane environment significantly influences ATP synthase assembly and function.

How does ATP binding affect the conformational dynamics of Koribacter versatilis ATP synthase complexes?

ATP binding induces significant conformational changes in the ATP synthase complex, which can be analyzed using several complementary approaches:

Conformational changes upon ATP binding:

• In the catalytic sites:

  • ATP binding triggers the transition from "open" to "closed" conformations in β subunits

  • Different rotational states show distinct positions of the rotor (subunits γεc₁₀)

  • The symmetry mismatch between the F₁ and F₀ sectors results in sequential conformational changes (3, 4, and 3 c-subunit steps)

• In the peripheral stalk (including atpF):

  • The C-terminal water-soluble part of subunit b displays significant conformational variability between states

  • ATP binding influences the flexibility of the peripheral stalk

  • These conformational changes help maintain efficient energy coupling

• In the membrane domain:

  • ATP binding affects the interaction between subunit a and the c-ring

  • These changes are crucial for proton translocation through the membrane

Research on bacterial ATP synthases shows that "the structure of the yeast ATP synthase F₀ dimer, which lacked the F₁ region and an intact peripheral stalk, showed that the c-ring and subunit a are held together by hydrophobic interactions rather than by the peripheral stalk" . This suggests that ATP binding may regulate these hydrophobic interactions through allosteric effects.

What methodological approaches are recommended for studying the role of Koribacter versatilis atpF in acidic environmental adaptations?

Studying Koribacter versatilis atpF's role in acidic environmental adaptations requires specialized methodological approaches:

Environmental adaptation study methods:

• Comparative genomics and evolutionary analyses:

  • Analyze atpF sequence conservation across Acidobacteria from different pH environments

  • Identify signature residues associated with acid tolerance

  • Compare with atpF sequences from neutrophilic bacteria

• Functional characterization under controlled pH conditions:

  • Measure ATP synthesis/hydrolysis activities across pH gradients (pH 3-7)

  • Assess proton pumping efficiency using fluorescent probes

  • Determine pH-dependent conformational changes using spectroscopic methods

• Mutagenesis approaches:

  • Generate site-directed mutants targeting charged residues in atpF

  • Create chimeric proteins with atpF regions from non-acidophilic bacteria

  • Assess functional consequences in heterologous expression systems

• In situ environmental studies:

  • Use metatranscriptomic analysis to measure atpF expression in natural acidic environments

  • Employ environmental proteomics to assess post-translational modifications

  • Implement stable isotope probing to track ATP synthase activity in situ

Research on Acidobacteria in peatland environments has shown that members of this phylum have evolved specific adaptations for acidic conditions. Studies have demonstrated that "Acidobacteria with a dissimilatory sulfur metabolism impact organic matter decomposition in wetlands" , suggesting that ATP synthesis in these environments may have unique adaptations. The genomic analysis of Acidobacteria has revealed "traits for desiccation resistance, biofilm formation, and/or contribution to soil structure" , indicating that ATP synthase components likely play important roles in these adaptation mechanisms.

What are the best approaches for analyzing potential post-translational modifications of Koribacter versatilis atpF?

Post-translational modifications (PTMs) of atpF can significantly impact ATP synthase function. The following analytical approaches are recommended:

PTM analysis methodology:

• Mass spectrometry-based approaches:

  • High-resolution LC-MS/MS for comprehensive PTM mapping

  • Targeted multiple reaction monitoring (MRM) for quantitative PTM analysis

  • Electron transfer dissociation (ETD) for labile modification identification

• Site-specific mutagenesis:

  • Mutate putative modification sites to non-modifiable residues

  • Create phosphomimetic mutations (e.g., Ser/Thr → Asp/Glu)

  • Assess functional consequences of mutation

• Specific PTM detection methods:

  • Phosphorylation: Pro-Q Diamond staining, phospho-specific antibodies

  • Glycosylation: Periodic acid-Schiff staining, lectin affinity

  • Acetylation: Acetylation-specific antibodies

• Temporal dynamics of PTMs:

  • Pulse-chase experiments with isotopically labeled precursors

  • Time-course analysis following environmental stress

  • Correlation of PTM patterns with ATP synthase activity

While specific information on PTMs of Koribacter versatilis atpF is limited, research on bacterial ATP synthases indicates that phosphorylation and acetylation can regulate enzymatic activity. Analysis of the atpF sequence reveals multiple potential modification sites, including serine/threonine residues for phosphorylation and lysine residues for acetylation or ubiquitination.

How can researchers effectively design experiments to investigate the nucleotide binding properties of Koribacter versatilis ATP synthase complexes?

Designing experiments to investigate nucleotide binding properties requires careful consideration of multiple factors:

Experimental design framework:

• Purification strategies:

  • For intact ATP synthase complexes: Gentle detergent solubilization followed by affinity chromatography

  • For subcomplex analysis: Targeted expression of relevant subunits with appropriate tags

  • For atpF-specific studies: Recombinant expression with N-terminal His-tag

• Nucleotide binding assays:

  • Isothermal titration calorimetry (ITC) for determining binding affinities and thermodynamic parameters

  • Microscale thermophoresis for measuring binding under various conditions

  • Fluorescence-based assays using fluorescent ATP analogs

• ATPase activity measurements:

  • Spectrophotometric assays coupling ATP hydrolysis to NADH oxidation

  • Measurement of inorganic phosphate release using colorimetric methods

  • Monitoring ATP-dependent proton pumping in reconstituted proteoliposomes

• Nucleotide specificity studies:

  • Compare binding and hydrolysis of different nucleotides (ATP, GTP, CTP)

  • Analyze the effects of divalent cations (Mg²⁺, Ca²⁺, Mn²⁺)

  • Investigate competitive inhibition with non-hydrolyzable analogs

Research indicates that bacterial ATP synthases can utilize different nucleotides as cofactors. For example, "BceSIV activity is strongly stimulated by the addition of cofactor ATP or GTP" , suggesting that similar nucleotide preferences might be observed in Koribacter versatilis ATP synthase. Furthermore, ATPase and GTPase assays can be effectively used to quantify nucleotide hydrolysis activities: "Reactions were carried out in a UV-transparent clear-bottom 96-well plate at 37°C for 12 min, with data collected every 10 s. The optical density at 340 nm (OD₃₄₀) was read" .