Recombinant Nitrosomonas europaea ATP synthase subunit delta (atpH)

What is the ATP synthase subunit delta (atpH) in Nitrosomonas europaea?

The ATP synthase subunit delta in Nitrosomonas europaea is a component of the F-type ATPase complex, specifically part of the F1 sector. This protein is encoded by the atpH gene (also designated as NE0203) and functions as a critical element in the energy production machinery of this chemolithoautotrophic ammonia-oxidizing bacterium. The protein spans 178 amino acids in length and serves as part of the central stalk that connects the F1 and F0 sectors, participating in the rotational mechanism that couples proton translocation to ATP synthesis .

What is the complete amino acid sequence of Nitrosomonas europaea ATP synthase subunit delta?

The full amino acid sequence of the ATP synthase subunit delta from Nitrosomonas europaea (strain ATCC 19718 / NBRC 14298) consists of 178 amino acids as follows:

MAEAITIARP YAEAVFKLAR ESGSLFSWSE TLDAVNSIVR ESQIRELISN PLISSVKLRE IIFSVCGKKL NEDGKRLVSL LIDNQRLLVM PQIHELFEQL KAQHESILEA EVVSAFPLDS GQLEKLVSIL EAKFQRKVKA EVSVDSELIG GVRIKIGDQV VDSSVHGKLE AMATALKS

This sequence is critical for understanding protein structure-function relationships and for designing expression constructs for recombinant production.

How does ATP synthase subunit delta relate to energy metabolism in N. europaea?

The ATP synthase subunit delta plays a vital role in the unique energy metabolism of Nitrosomonas europaea. As a chemolithoautotrophic ammonia-oxidizing bacterium, N. europaea derives energy from the oxidation of ammonia to nitrite. Cell-free preparations of N. europaea can oxidize hydroxylamine (but not ammonium ion) to nitrite, with the ATP synthase complex capturing the energy from this process .

Studies have demonstrated that during hydroxylamine oxidation, P32-labeled inorganic phosphate is incorporated into organic fractions, including ATP and ADP, indicating the direct coupling between the oxidation pathway and ATP synthesis . The delta subunit specifically helps maintain the structural and functional integrity of the ATP synthase complex, ensuring efficient energy conversion from the proton gradient to ATP formation. This energy production system is essential for supporting N. europaea's autotrophic lifestyle, where all cellular carbon is derived from CO2 fixation.

Which expression systems are available for recombinant production of N. europaea ATP synthase subunit delta?

Multiple expression systems have been successfully employed for the recombinant production of Nitrosomonas europaea ATP synthase subunit delta. Each system offers distinct advantages and considerations:

The choice of expression system should be guided by specific research requirements, including need for post-translational modifications, protein solubility considerations, and functional integrity requirements .

What is the significance of in vivo biotinylation using AviTag-BirA technology for this protein?

In vivo biotinylation using AviTag-BirA technology offers significant advantages for researchers working with recombinant Nitrosomonas europaea ATP synthase subunit delta:

The E. coli biotin ligase (BirA) specifically attaches biotin covalently to the 15 amino acid AviTag peptide sequence. This process occurs through an amide linkage between biotin and a specific lysine residue within the AviTag . This biotinylation approach is valuable because:

• It provides consistent, site-specific labeling at a predetermined location on the protein.

• The biotin-streptavidin interaction is extremely strong (Kd ≈ 10^-15 M), enabling robust detection and immobilization applications.

• The biotinylation occurs during protein expression in E. coli, eliminating the need for post-purification chemical modification.

• It allows for oriented immobilization of the protein for structural studies or interaction analyses.

• The approach minimizes structural disruption compared to chemical biotinylation methods.

For structural and functional studies of ATP synthase components, this technology enables precise control over protein orientation and attachment, facilitating experimental reproducibility.

How can researchers optimize expression conditions for maximum protein yield?

Optimizing expression conditions for recombinant Nitrosomonas europaea ATP synthase subunit delta requires systematic evaluation of multiple parameters. Based on established methodologies for similar proteins, researchers should consider:

• Induction parameters optimization:

  • IPTG concentration (for bacterial systems): Testing ranges from 0.1-1.0 mM

  • Temperature: Evaluating lower temperatures (15-25°C) that often improve proper folding

  • Induction timing: Inducing at different cell densities (OD600 0.4-0.8)

  • Duration: Testing expression periods from 4-24 hours

• Media composition factors:

  • Rich vs. minimal media effects on expression levels

  • Supplementation with trace elements that may be required for proper folding

  • Carbon source variations (glucose vs. glycerol)

• Statistical optimization approach:

  • Implementation of response surface methodology (RSM) based on central composite design (CCD)

  • Testing three levels for each factor (+1, 0, -1) representing high, middle, and low values

  • Using software such as Minitab for experimental design and analysis

A typical optimization experiment might use the following design matrix for three key factors:

This systematic approach allows researchers to identify optimal conditions while accounting for interaction effects between variables .

What purification strategies are most effective for recombinant N. europaea ATP synthase subunit delta?

Effective purification of recombinant Nitrosomonas europaea ATP synthase subunit delta requires strategic selection of techniques based on expression system and protein characteristics. A recommended multi-step purification process includes:

• Initial capture:

  • For tagged constructs: Immobilized metal affinity chromatography (IMAC) using nickel or cobalt resins

  • For AviTag-biotinylated protein: Streptavidin affinity chromatography

  • For untagged protein: Ion exchange chromatography (typically anion exchange at pH 7.5-8.0)

• Intermediate purification:

  • Size exclusion chromatography to separate monomeric protein from aggregates

  • Hydrophobic interaction chromatography as an orthogonal step

• Polishing:

  • High-resolution ion exchange chromatography

  • Hydroxyapatite chromatography for removal of endotoxins (for E. coli-expressed proteins)

During purification, maintaining appropriate pH is critical, as research with other bacterial proteins shows significant pH dependence. For instance, inactivation kinetics studies on N. europaea demonstrate that pH values between 7 and 9 can significantly affect protein stability, with pH 7 often providing optimal stability for many bacterial proteins .

The target purity should be >85% as assessed by SDS-PAGE, which is standard for research applications of recombinant proteins .

How can researchers assess the structural integrity of purified recombinant protein?

A comprehensive assessment of structural integrity for purified recombinant Nitrosomonas europaea ATP synthase subunit delta should employ multiple complementary techniques:

• Primary structure verification:

  • Mass spectrometry to confirm molecular weight

  • N-terminal sequencing to verify the correct start of the protein

  • Peptide mapping to ensure complete coverage of the sequence

• Secondary and tertiary structure analysis:

  • Circular dichroism (CD) spectroscopy to determine secondary structure content

  • Fluorescence spectroscopy to evaluate the tertiary structure environment of tryptophan residues

  • Fourier-transform infrared spectroscopy (FTIR) for additional structural information

• Stability assessment:

  • Differential scanning calorimetry (DSC) to determine thermal transitions

  • pH stability profile determination

  • Long-term storage stability testing at different temperatures

• Functional verification:

  • Interaction studies with other ATP synthase subunits

  • ATPase activity assays if applicable

  • Binding studies with known ligands

For proper quality control, researchers should compare the spectroscopic profiles of the recombinant protein with theoretical predictions based on the amino acid sequence of the ATP synthase delta subunit . Deviations from expected profiles may indicate folding issues or structural perturbations.

What are the critical pH considerations when working with recombinant N. europaea proteins?

pH considerations are critical when working with recombinant Nitrosomonas europaea ATP synthase subunit delta due to their significant impact on protein stability, activity, and interactions. Key considerations include:

• Buffer selection based on pH ranges:

  • pH 1-3.5: Glycine-HCl buffers

  • pH 3.5-6: Sodium acetate-acetic acid buffers

  • pH 6-7: Tris-acetic acid buffers

  • pH 7-9: Tris-HCl buffers

  • pH 9-10: Glycine-NaOH buffers

• Physiological relevance:

  • N. europaea naturally grows in environments with pH ranging from approximately 7.4 to 7.8

  • Studies show that when the pH of N. europaea cultures decreases below approximately 7.4 during growth, significant metabolic changes occur, including alterations in polyphosphate accumulation and rates of nitrite synthesis

• pH effects on protein characteristics:

  • Stability: The ATP synthase components typically show optimal stability in the pH range of 7.0-8.5

  • Solubility: pH-dependent solubility profiles should be determined empirically

  • Activity: Enzymatic activity may show distinct pH optima that differ from stability optima

• pH considerations during purification:

  • When using imidazole for elution of His-tagged proteins, pH effects should be considered. At pH 5.8, approximately 95% of imidazole would be protonated, affecting its interaction with metal ions and protein

  • pH adjustments may be necessary after buffer exchange steps

Researchers should conduct pH stability profiles and activity assays across a relevant pH range (typically pH 6-9) to determine optimal conditions for their specific experimental goals.

How should researchers design experiments to study ATP synthase delta subunit interactions?

Designing robust experiments to study interactions involving the ATP synthase delta subunit from Nitrosomonas europaea requires a multi-faceted approach:

• Protein-protein interaction methodologies:

  • Pull-down assays using recombinant proteins with different affinity tags

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

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Microscale thermophoresis (MST) for solution-based interaction measurements

• Experimental design considerations:

  • Positive controls: Include known interacting partners

  • Negative controls: Use non-related proteins of similar size/charge characteristics

  • Validation through multiple techniques: Confirm interactions using at least two independent methods

  • Concentration range testing: Perform experiments across physiologically relevant concentrations

• Structural elements investigation:

  • Domain mapping through truncation constructs

  • Site-directed mutagenesis of predicted interface residues

  • Cross-linking studies to capture transient interactions

  • Hydrogen-deuterium exchange mass spectrometry to identify interaction surfaces

• Functional correlation:

  • Correlation of binding affinity with functional outcomes

  • Measurement of ATP synthesis/hydrolysis in reconstituted systems

  • Assessment of the impact of mutations on both binding and function

When designing these experiments, researchers should account for the natural operating environment of the ATP synthase complex. For N. europaea, this includes consideration of the physiological pH range (7.4-7.8) and temperature (25-30°C) that reflect the organism's native growth conditions .

What controls are essential when working with recombinant N. europaea ATP synthase proteins?

When working with recombinant Nitrosomonas europaea ATP synthase subunit delta, implementing appropriate controls is critical for experimental validity:

• Expression and purification controls:

  • Empty vector control: Expression host transformed with the vector lacking the insert

  • Non-recombinant host cells: Wild-type expression host without any plasmid

  • Non-related protein control: Expression of an unrelated protein using the same vector and conditions

• Functional and structural controls:

  • Heat-denatured protein: Same recombinant protein subjected to thermal denaturation

  • Related protein variant: A closely related ATP synthase subunit from another organism

  • Native protein preparation (if available): Naturally isolated protein from N. europaea

• Assay-specific controls:

  • Enzyme activity assays: Substrate-only and enzyme-only reactions

  • Binding studies: Known binders and non-binders to verify assay functionality

  • Structural analyses: Reference proteins with well-characterized structural profiles

• Environmental condition controls:

  • pH series testing: Parallel experiments at multiple pH values

  • Temperature variation: Testing at physiologically relevant versus non-optimal temperatures

  • Buffer component testing: Evaluation of specific buffer effects independent of pH

• Statistical and experimental design controls:

  • Technical replicates: Multiple measurements of the same sample

  • Biological replicates: Independent preparations of the recombinant protein

  • Randomization of sample order: Minimization of systematic errors

Published research with N. europaea emphasizes the importance of assessing culture purity and protein activity through routine monitoring, including microscopic examination, fluorescent in situ hybridization analyses, and activity measurements .

How can researchers optimize temperature conditions for experiments with this protein?

Temperature optimization for experiments involving recombinant Nitrosomonas europaea ATP synthase subunit delta requires systematic evaluation of multiple aspects:

• Temperature effects on protein stability:

  • Perform thermal shift assays (differential scanning fluorimetry) to determine melting temperature (Tm)

  • Conduct time-course stability studies at different temperatures (4°C, 25°C, 37°C)

  • Evaluate freeze-thaw stability for storage considerations

• Temperature impacts on functional assays:

  • Test enzymatic activity across a temperature range (typically 15-40°C)

  • Determine if temperature optima for activity differ from those for stability

  • Consider that N. europaea naturally grows at moderate temperatures (optimal around 25-30°C)

• Temperature optimization experimental design:

  • Initial broad-range screening (15-40°C in 5°C increments)

  • Fine-tuning around identified optimal ranges (1-2°C increments)

  • Integration with other parameters (pH, buffer composition) using response surface methodology

• Practical temperature considerations:

  • Implement precise temperature control using water baths or thermostated instruments

  • Account for temperature gradients in larger reaction vessels

  • Monitor actual sample temperatures rather than ambient or set temperatures

Research on N. europaea has demonstrated that the organism is typically cultivated at approximately a temperature of 26°C, with experiments conducted at 28°C . This suggests that protein components from this organism may be optimally stable and functional around this temperature range, which should serve as a starting point for optimization experiments.

How should researchers analyze variability in recombinant protein expression across different batches?

Analyzing batch-to-batch variability in recombinant Nitrosomonas europaea ATP synthase subunit delta expression requires systematic approaches to ensure data reliability:

• Quantitative assessment methods:

  • Densitometric analysis of SDS-PAGE gels (normalized to standards)

  • Protein concentration determination using multiple methods (Bradford, BCA, absorbance at 280 nm)

  • Activity assays normalized to protein concentration

  • Mass spectrometry-based absolute quantification

• Statistical approaches for batch comparison:

  • Analysis of variance (ANOVA) to identify significant batch effects

  • Mixed-effects models incorporating batch as a random effect

  • Coefficient of variation (CV) calculation across batches to quantify variability

  • Control charts to monitor process stability over time

• Normalization strategies:

  • Reference standards included in each batch analysis

  • Internal controls measured alongside experimental samples

  • Batch-specific calibration curves for activity measurements

  • Ratio metrics comparing target protein to total protein

• Experimental design to address batch variability:

  • Inclusion of multiple batches within each experiment when possible

  • Randomized block designs with batch as a blocking factor

  • Stratified sampling across batches for downstream applications

Researchers should establish acceptable variability thresholds based on their experimental requirements. For most applications, maintaining batch-to-batch protein yield variations below 15-20% and functional activity variations below 10-15% is considered good practice, though more stringent criteria may be needed for specific applications .

How do you reconcile differences in activity between native and recombinant forms of the protein?

Reconciling activity differences between native and recombinant forms of Nitrosomonas europaea ATP synthase subunit delta requires systematic analysis:

• Potential sources of difference:

  • Post-translational modifications present in native but not recombinant protein

  • Structural variations due to expression host environments

  • Effects of affinity tags or fusion partners in recombinant constructs

  • Differences in protein-protein interactions when isolated versus in complex

• Analytical comparison approaches:

  • Side-by-side activity assays under identical conditions

  • Structural analysis using circular dichroism and fluorescence spectroscopy

  • Mass spectrometry to identify chemical differences

  • Kinetic parameter determination (Km, Vmax) for both forms

• Reconciliation strategies:

  • Correction factors based on empirical comparison data

  • Expression in multiple systems to identify host-specific effects

  • Tag removal and re-assessment of activity

  • Reconstitution with other ATP synthase components

• Interpretation framework:

  • Consider the natural context of native protein function

  • Evaluate isolation procedures' impact on native protein activity

  • Account for differences in protein quantification methods

  • Assess the influence of the complete ATP synthase complex

Studies with related proteins have shown that native N. europaea enzymes may exhibit different characteristics based on growth conditions. For example, when the pH of a culture of Nitrosomonas decreases during growth below approximately 7.4, significant changes in enzyme activity can occur . Such environmental factors should be considered when comparing native and recombinant protein activities.

What statistical methods are most appropriate for analyzing functional studies of this protein?

Selecting appropriate statistical methods for analyzing functional studies of recombinant Nitrosomonas europaea ATP synthase subunit delta enhances data reliability and interpretability:

• Experimental design considerations:

  • Power analysis to determine sample size requirements

  • Randomized complete block designs to control for batch effects

  • Factorial designs for multi-parameter investigations

  • Latin square designs to minimize confounding variables

• Basic statistical analyses:

  • Descriptive statistics (mean, median, standard deviation, confidence intervals)

  • Normality testing prior to parametric analysis (Shapiro-Wilk test)

  • Student's t-test for comparing two conditions

  • ANOVA with post-hoc tests (Tukey's HSD, Bonferroni) for multiple comparisons

• Advanced statistical approaches:

  • Multiple regression for modeling relationships between variables

  • Response surface methodology for optimization experiments

  • Principal component analysis for multivariate data reduction

  • Mixed-effects models to account for random and fixed effects

• Specialized methods for kinetic and binding data:

  • Non-linear regression for enzyme kinetics (Michaelis-Menten, Hill equation)

  • Global fitting for complex binding models

  • Analysis of time-series data with repeated measures ANOVA

When analyzing data from enzyme activity assays, researchers should consider models appropriate to the specific type of activity being measured. For instance, studies on L-Malate dehydrogenase from N. europaea found that the enzyme exhibited simple Michaelis-Menten kinetics with a Km for oxaloacetate of 20 μM and a Km for NADH of 22 μM . Similar kinetic analyses may be appropriate for ATP synthase components depending on the specific activity being measured.

What are common challenges in expressing N. europaea ATP synthase subunit delta and their solutions?

Researchers frequently encounter specific challenges when expressing recombinant Nitrosomonas europaea ATP synthase subunit delta, each requiring targeted solutions:

Studies with bacterial proteins similar to ATP synthase components have shown that maintaining appropriate pH is crucial during expression and purification. For N. europaea proteins, this typically means keeping conditions in the pH 7.0-8.5 range, which supports both stability and activity .

How can researchers overcome solubility issues with this recombinant protein?

Addressing solubility challenges with recombinant Nitrosomonas europaea ATP synthase subunit delta requires a multi-faceted approach:

• Expression condition modifications:

  • Reduce expression temperature to 15-20°C to slow folding kinetics

  • Decrease inducer concentration to lower expression rate

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Use specialized strains designed for difficult-to-express proteins

• Construct engineering strategies:

  • Fusion with solubility-enhancing tags (MBP, SUMO, GST)

  • Expression of individual domains if full-length protein is problematic

  • Removal of predicted aggregation-prone regions if non-essential

  • Site-directed mutagenesis of problematic residues

• Buffer optimization:

  • Systematic pH screening (typically in 7.0-8.5 range for N. europaea proteins)

  • Addition of stabilizing agents:

    • Osmolytes (glycerol 5-15%, sucrose, trehalose)

    • Amino acids (arginine, proline)

    • Mild non-ionic detergents (0.01-0.05% Triton X-100)

  • Increased ionic strength (150-300 mM NaCl)

• Refolding approaches if inclusion bodies form:

  • Gradual dialysis from denaturing conditions

  • On-column refolding during purification

  • Pulse dilution into refolding buffer

  • Use of artificial chaperones (cyclodextrin-detergent systems)

Research with ATP synthase components and other bacterial proteins suggests that maintaining conditions similar to the organism's natural environment often supports better solubility. For N. europaea, which grows optimally at pH 7.4-7.8 and temperatures around 25-30°C, these parameters serve as a good starting point for solubility optimization .

What strategies ensure long-term stability of purified recombinant protein?

Ensuring long-term stability of purified recombinant Nitrosomonas europaea ATP synthase subunit delta requires careful consideration of storage conditions and stabilizing factors:

• Storage format optimization:

  • Lyophilized powder format for maximum stability

  • Liquid solutions with appropriate stabilizers

  • Flash-frozen aliquots at -80°C to minimize freeze-thaw cycles

• Buffer composition for optimal stability:

  • Optimize pH based on stability profiles (typically pH 7.0-8.5 for N. europaea proteins)

  • Include stabilizing agents:

    • Glycerol (10-25%) as cryoprotectant

    • Reducing agents if cysteine residues are present

    • Specific metal ions if required for structural integrity

  • Appropriate salt concentration (typically 100-200 mM NaCl)

• Storage condition recommendations:

  • Temperature guidelines:

    • -80°C for long-term storage (>6 months)

    • -20°C for medium-term storage (1-6 months)

    • 4°C for short-term use (days to weeks, depending on stability)

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Protect from light if photosensitive residues are present

• Stability monitoring protocol:

  • Regular activity testing of stored samples

  • Size-exclusion chromatography to detect aggregation

  • SDS-PAGE to assess degradation

  • Functional assays specific to the protein's role

For ATP synthase components, maintaining reducing conditions may be important if the protein contains cysteine residues that could form inappropriate disulfide bonds. Additionally, considering that N. europaea proteins function in specific environmental conditions, mimicking these conditions (appropriate pH, ionic strength) during storage can help maintain native-like structure and function .