Recombinant Nitratiruptor sp. ATP synthase subunit beta (atpD)

What is the structure and function of Nitratiruptor sp. ATP synthase subunit beta?

ATP synthase subunit beta, encoded by the atpD gene in Nitratiruptor sp., is a critical component of the F₁ catalytic domain of ATP synthase. This subunit forms part of the α₃β₃ hexamer in the hydrophilic F₁ subcomplex, which together with the hydrophobic FO subcomplex constitutes the complete ATP synthase holoenzyme . The beta subunit contains the catalytic sites responsible for ATP synthesis and hydrolysis, utilizing the proton gradient generated across the membrane during cellular respiration.

The subunit beta participates in the rotary mechanism that couples proton translocation to ATP synthesis through conformational changes in the enzyme complex. Similar to other ATP synthases, such as those found in A. baumannii, the Nitratiruptor sp. ATP synthase likely has a conserved architecture where three beta subunits alternate with three alpha subunits in a hexameric ring around the central γ subunit .

Why is recombinant expression of ATP synthase subunits important in research?

Recombinant expression of ATP synthase subunits provides several critical advantages for research:

• Protein availability: Natural abundance of ATP synthase in native sources is often limited, making recombinant expression essential for obtaining sufficient quantities for biochemical and structural studies.

• Molecular manipulation: Recombinant DNA technology allows for the introduction of mutations, truncations, and fusion tags, enabling structure-function relationship studies. For example, researchers have used recombinant systems to study single amino acid substitutions in ATP synthase subunits to understand regulatory mechanisms .

• System simplification: Expression of individual subunits or subcomplexes helps dissect the roles of specific components. As demonstrated with A. baumannii F₁-ATPase, researchers can generate and purify recombinant subcomplexes (α₃:β₃:γ:ε) to study specific aspects like latent ATP hydrolysis .

• Comparative studies: Recombinant expression facilitates comparative analyses between ATP synthases from different species, helping to understand evolutionary adaptations and conserved mechanisms across bacterial, chloroplast, and mitochondrial varieties .

How do auxiliary ATP binding sites in recombinant ATP synthases affect enzyme kinetics?

Auxiliary ATP binding sites can significantly impact the kinetics and efficiency of ATP synthases, as demonstrated in studies with other ATP-dependent enzymes like RecBCD. These secondary binding sites typically exhibit different affinities and chemical interactions compared to the canonical catalytic sites .

Key characteristics of auxiliary binding sites include:

• Biphasic binding behavior: Equilibrium binding assays may reveal a biphasic pattern as a function of nucleotide concentration, indicating the presence of both high-affinity (strong) and low-affinity (weak) binding sites .

• Distinct chemical interactions: While catalytic sites interact strongly with both the phosphate groups and base moieties of ATP, auxiliary sites may interact primarily through the base and sugar portions of the nucleotide .

• Differential sensitivity to conditions: Auxiliary sites often show distinct responses to salt concentrations and nucleotide analogs compared to catalytic sites .

What are the challenges in expressing the complete ATP synthase complex versus individual subunits?

Expressing complete ATP synthase complexes presents significantly greater challenges than expressing individual subunits:

What are the optimal expression systems for recombinant Nitratiruptor sp. ATP synthase subunit beta?

The choice of expression system for recombinant Nitratiruptor sp. ATP synthase subunit beta should consider several factors:

• E. coli-based systems: Most commonly used for initial attempts due to:

  • Rapid growth and high yield

  • Well-established protocols for protein expression

  • Availability of various fusion tags and expression vectors

  • Successful precedent with other bacterial ATP synthase subunits

• Considerations for optimization:

  • Codon optimization for the atpD gene sequence

  • Reduced expression temperature (16-20°C) to improve folding

  • Use of specialized E. coli strains (e.g., C41/C43) designed for membrane protein expression

  • IPTG concentration titration for optimal induction

• Alternative systems:

  • Cell-free expression systems for rapid screening

  • Bacillus subtilis for better expression of some bacterial proteins

  • Yeast systems for complex proteins requiring eukaryotic folding machinery

For the beta subunit specifically, which is water-soluble as part of the F₁ complex, E. coli expression systems have proven successful for similar proteins and would likely be appropriate for the Nitratiruptor sp. variant as well .

What purification strategies are most effective for obtaining active recombinant ATP synthase beta subunit?

Effective purification of recombinant Nitratiruptor sp. ATP synthase beta subunit requires a multi-step approach:

• Initial affinity purification:

  • His-tag affinity chromatography using Ni-NTA or TALON resins

  • Alternative tags such as GST or MBP may improve solubility

  • Optimal buffer conditions typically include 20-50 mM Tris-HCl pH 7.5-8.0, 100-300 mM NaCl

• Secondary purification steps:

  • Ion exchange chromatography (MonoQ) for removing impurities

  • Size exclusion chromatography for isolating properly folded monomeric protein

  • ATP-agarose affinity chromatography to select for functional protein capable of nucleotide binding

• Quality assessment:

  • SDS-PAGE for purity verification

  • Western blotting for identity confirmation

  • Circular dichroism for secondary structure assessment

  • Thermal shift assays for stability evaluation

For maintaining activity during purification, it's critical to include stabilizing agents such as glycerol (10-20%), reducing agents (1-5 mM DTT or TCEP), and potentially nucleotides (ATP or ADP) throughout the purification process .

How can one assess the ATPase activity of recombinant Nitratiruptor sp. ATP synthase beta subunit?

Assessment of ATPase activity can be performed using several complementary methods:

• In-gel ATPase activity assay:

  • A well-established method previously applied to mitochondrial, chloroplast, and cyanobacterial ATP synthases

  • Samples are run on native PAGE gels and incubated in a reaction mixture containing ATP and divalent cations

  • Activity is visualized by the formation of precipitates

  • This method can detect activity after very short incubation periods (as short as 0.5 h)

• Spectrophotometric coupled enzyme assays:

  • ATP hydrolysis coupled to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Continuous monitoring of NADH absorbance decrease at 340 nm

  • Allows for real-time kinetic measurements and inhibitor studies

• Malachite green phosphate assay:

  • Direct measurement of inorganic phosphate released during ATP hydrolysis

  • End-point assay with high sensitivity

  • Suitable for high-throughput screening

• Radiolabeled ATP hydrolysis:

  • Using [γ-³²P]ATP to monitor release of ³²P-labeled inorganic phosphate

  • Higher sensitivity than colorimetric methods

  • Allows detection of very low activity levels

For the recombinant Nitratiruptor sp. beta subunit, the choice of detergent can significantly impact activity. Mild detergents like LDAO generally preserve higher activity levels compared to harsher detergents like TODC .

What approaches can be used to study nucleotide binding to recombinant ATP synthase beta subunit?

Several complementary approaches can be employed to characterize nucleotide binding:

• Fluorescence-based methods:

  • Förster Resonance Energy Transfer (FRET) between protein tryptophans and fluorescent nucleotide analogs (e.g., mantADP, mantAMPpNp)

  • Enables determination of binding affinities and detection of multiple binding sites with different affinities

  • Can reveal biphasic binding behavior indicative of distinct binding sites

• Equilibrium binding assays:

  • Isothermal titration calorimetry (ITC) for direct measurement of binding thermodynamics

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

  • Microscale thermophoresis (MST) for binding in solution with minimal protein consumption

• Time-resolved kinetics:

  • Stopped-flow spectroscopy to monitor rapid binding events

  • Quench-flow techniques for measuring fast chemical steps

  • Temperature adjustment (e.g., 6°C) can slow down reactions to facilitate monitoring of transient kinetics

• Competitive binding studies:

  • Using unmodified nucleotides to compete with fluorescent analogs

  • Reveals differences in binding site preferences and affinities

  • Can distinguish between catalytic and auxiliary binding sites

• Structural mapping:

  • Nucleotide cross-linking followed by mass spectrometry

  • Computational docking and molecular dynamics simulations

  • Site-directed mutagenesis of predicted binding residues

How can researchers troubleshoot low expression or activity of recombinant Nitratiruptor sp. ATP synthase beta subunit?

When facing challenges with expression or activity, systematically address these common issues:

Additionally, researchers should consider whether the beta subunit alone is expected to show activity or if it requires association with other subunits. For ATP synthase beta subunits, full activity often requires assembly with alpha subunits to form the F₁ complex .

What strategies can be employed when encountering data contradictions in ATP binding and hydrolysis experiments?

When facing contradictory data in ATP binding and hydrolysis experiments:

• Validate experimental conditions:

  • Ensure proper temperature and pH control across experiments

  • Verify buffer composition consistency, particularly divalent cation concentrations

  • Check for contaminating ATPase activity in reagents

• Consider regulatory mechanisms:

  • Latent ATPase activity may be observed, requiring activation conditions

  • Inhibitory subunits may be present (e.g., subunit ε in A. baumannii F₁-ATPase)

  • Allosteric regulation by nucleotides at auxiliary binding sites might occur

• Examine methodological differences:

  • Different detection methods may have varying sensitivities

  • Some assays may be influenced by specific buffer components

  • Detergent effects can significantly impact activity measurements

• Investigate kinetic complexities:

  • ATP synthases often exhibit biphasic behavior in nucleotide binding

  • Multiple binding sites with different affinities can complicate interpretations

  • Cooperativity between subunits may lead to non-linear kinetics

• Sample heterogeneity:

  • Verify protein purity and homogeneity via size exclusion chromatography

  • Assess oligomeric state as this can affect activity measurements

  • Consider using additional purification steps like MonoQ chromatography

When contradictions persist, parallel experiments with control proteins (e.g., well-characterized ATP synthases from E. coli or thermophilic bacteria) can provide helpful reference points.