Recombinant Bacillus thuringiensis subsp. konkukian ATP synthase subunit delta (atpH)

What is the molecular structure of B. thuringiensis subsp. konkukian ATP synthase subunit delta?

The ATP synthase subunit delta (atpH) in Bacillus thuringiensis subsp. konkukian is a protein that functions as a component of the stalk connecting the F₀ and F₁ domains of the ATP synthase complex. According to structural modeling data, the protein consists of 180 amino acids with the following sequence: MSNGIVAKRY AVALFKIAKE KHVLEMFEEE LRLVQNVYEK NGELHSFLTQ PNISKEQKKT FLANVFGSVS ESILNTLYIL IDNKRIDILS DIANEYVVLA NEERNVADAT VYSTRLLSEE EKLNIAEAFA KRTGKDAIRV KNVVDEDLLG GIKVRIGNRI YDGSLQGKLA RIQRELMKNR.

The computed structure model of this protein (available as AF_AFQ6HAX6F1 in the RCSB Protein Data Bank) has been generated using AlphaFold, with a global pLDDT (predicted Local Distance Difference Test) score of 84.29, indicating a relatively confident prediction of the protein's tertiary structure . The model was released in the AlphaFold database on December 9, 2021, and last modified on September 30, 2022 .

What role does ATP synthase subunit delta play in bacterial physiology?

ATP synthase subunit delta serves as a critical component of the ATP synthase complex, which synthesizes ATP from ADP using a proton or sodium gradient. The delta subunit specifically contributes to the structural integrity of the complex by linking the membrane-embedded F₀ domain with the catalytic F₁ domain. This linkage facilitates the transmission of conformational changes between the domains and may participate in the proton conduction process.

In bacterial energy metabolism, this subunit helps maintain the proper assembly and function of the entire ATP synthase complex, which is essential for cellular energy production. Disruption of the delta subunit function could potentially impair ATP synthesis and subsequently affect numerous cellular processes dependent on ATP availability.

What expression systems are most suitable for producing recombinant B. thuringiensis ATP synthase subunit delta?

For the recombinant expression of B. thuringiensis subsp. konkukian ATP synthase subunit delta, several expression systems can be employed based on research needs:

E. coli-based expression systems:

• BL21(DE3) strains are commonly used for expressing bacterial proteins due to their reduced protease activity

• pET vector systems under the control of T7 promoter provide high-yield expression

• Fusion tags such as His6, GST, or MBP can facilitate purification and potentially enhance solubility

Methodology for optimization:

• Clone the atpH gene (UniProt: Q6HAX6) into an expression vector with an appropriate fusion tag

• Transform into competent E. coli cells

• Screen multiple expression conditions varying:

  • Induction temperature (15-37°C)

  • IPTG concentration (0.1-1.0 mM)

  • Induction time (3-24 hours)

• Analyze expression levels using SDS-PAGE and western blotting

When working with ATP synthase components, researchers should consider that these proteins typically function as part of a complex, and isolated subunits may exhibit different folding characteristics compared to their native environment.

What purification strategies yield the highest purity of recombinant ATP synthase subunit delta?

A multi-step purification strategy is recommended for obtaining high-purity recombinant ATP synthase subunit delta:

Initial capture:

• For His-tagged constructs: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resin

• For GST-fusion proteins: Glutathione-Sepharose affinity chromatography

Intermediate purification:

• Ion exchange chromatography (considering the theoretical pI of the protein)

• Tag cleavage using TEV or thrombin protease (if tag removal is desired)

Polishing step:

• Size exclusion chromatography to remove aggregates and achieve high homogeneity

Buffer optimization considerations:

• pH range: 7.0-8.0 to maintain protein stability

• Salt concentration: 150-300 mM NaCl to prevent non-specific interactions

• Addition of glycerol (5-10%) to enhance protein stability

• Potential inclusion of reducing agents like DTT or β-mercaptoethanol (1-5 mM)

The purification protocol should be validated using SDS-PAGE, western blotting, and mass spectrometry to confirm identity and purity.

How reliable are computational structure predictions for ATP synthase subunit delta?

When evaluating computational model reliability, researchers should consider:

For ATP synthase subunit delta, researchers should note that computational models currently lack experimental validation, as indicated in the model metadata: "There are no experimental data to verify the accuracy of this computed structure model" . Therefore, while useful for generating hypotheses and designing experiments, these models should be interpreted cautiously until experimentally validated.

What experimental methods can validate or refine computational structure predictions?

Several experimental approaches can be employed to validate or refine the computational structure prediction of ATP synthase subunit delta:

X-ray crystallography:

• Provides high-resolution structural information

• Requires obtaining protein crystals, which can be challenging for membrane-associated proteins

• Method:

  • Express and purify protein to >95% homogeneity

  • Screen numerous crystallization conditions

  • Optimize crystal growth for diffraction quality

  • Collect and process diffraction data

  • Solve structure using molecular replacement with the computational model as a starting point

Nuclear Magnetic Resonance (NMR) spectroscopy:

• Useful for smaller proteins or domains (<25 kDa)

• Provides information about protein dynamics in solution

• Method:

  • Express isotopically labeled protein (¹⁵N, ¹³C)

  • Collect multi-dimensional NMR spectra

  • Assign resonances and calculate distance constraints

  • Generate and refine structural models

Cryo-electron microscopy (cryo-EM):

• Increasingly powerful for resolving protein structures

• Particularly valuable for large complexes like complete ATP synthase

• Method:

  • Prepare protein sample in vitrified ice

  • Collect micrographs under cryogenic conditions

  • Process images to generate 3D reconstructions

  • Fit atomic models into EM density maps

Integrative structural biology approaches:

• Combine multiple experimental techniques with computational models

• Particularly useful for challenging systems like membrane-associated proteins

• Example workflow:

  • Use computational models for initial hypothesis generation

  • Validate secondary structure elements with circular dichroism

  • Probe specific regions with hydrogen-deuterium exchange mass spectrometry

  • Identify domain interactions with crosslinking mass spectrometry

  • Integrate all data to refine the structural model

How can interactions between ATP synthase subunit delta and other complex components be studied?

Investigating interactions between ATP synthase subunit delta and other components of the ATP synthase complex requires specialized techniques:

Co-immunoprecipitation (Co-IP):

• Useful for identifying stable protein-protein interactions

• Method:

  • Generate antibodies against ATP synthase subunit delta or use tagged recombinant protein

  • Prepare bacterial cell lysate under non-denaturing conditions

  • Immunoprecipitate subunit delta with specific antibodies

  • Analyze co-precipitated proteins by mass spectrometry

Surface Plasmon Resonance (SPR):

• Provides quantitative binding kinetics data

• Method:

  • Immobilize purified ATP synthase subunit delta on a sensor chip

  • Flow potential interacting partners over the surface

  • Measure association and dissociation rates

  • Calculate binding affinity constants

Crosslinking coupled with mass spectrometry:

• Captures transient or weak interactions

• Provides spatial constraints for structural modeling

• Method:

  • Treat purified ATP synthase complex with crosslinking reagents

  • Digest crosslinked samples with proteases

  • Enrich crosslinked peptides

  • Identify crosslinks by mass spectrometry

  • Map interaction interfaces based on crosslinked residues

Bacterial two-hybrid assays:

• In vivo system for detecting protein interactions

• Method:

  • Clone ATP synthase subunit delta and potential interactors into appropriate vectors

  • Co-transform into reporter bacterial strain

  • Measure reporter gene activation as indicator of interaction

  • Validate with controls and competition assays

For studying the specific role of subunit delta in ATP synthase assembly, researchers can also employ mutagenesis approaches followed by functional assays to identify critical residues involved in complex formation and activity.

What methods can determine the contribution of ATP synthase subunit delta to enzyme function?

Site-directed mutagenesis:

• Allows systematic analysis of key residues

• Method:

  • Identify conserved or structurally important residues based on sequence alignments and structural models

  • Generate point mutations using PCR-based methods

  • Express and purify mutant proteins

  • Assess effects on ATP synthase assembly and activity

  • Compare kinetic parameters of wild-type vs. mutant complexes

Reconstitution experiments:

• Enables assessment of subunit delta's role in complex assembly

• Method:

  • Purify individual ATP synthase components

  • Assemble complexes with and without subunit delta

  • Measure ATP synthesis/hydrolysis activities

  • Analyze complex stability using native gel electrophoresis or analytical ultracentrifugation

ATP synthesis/hydrolysis assays:

• Quantifies functional impact of subunit delta modifications

• Method for ATP synthesis:

  • Reconstitute ATP synthase into liposomes

  • Generate proton gradient (pH or electrical potential)

  • Add ADP and Pi

  • Measure ATP production using luciferase assay

• Method for ATP hydrolysis:

  • Incubate purified ATP synthase with ATP

  • Measure inorganic phosphate release colorimetrically

  • Calculate enzymatic parameters (Km, Vmax)

Proton translocation measurements:

• Assesses coupling between proton movement and ATP synthesis

• Method:

  • Reconstitute ATP synthase into proteoliposomes

  • Include pH-sensitive fluorescent dyes

  • Initiate ATP hydrolysis and monitor pH changes

  • Compare efficiency with and without functional subunit delta

How conserved is ATP synthase subunit delta across bacterial species?

ATP synthase subunit delta shows varying degrees of conservation across bacterial species, which can provide insights into functional constraints and evolutionary relationships:

Sequence conservation analysis:

• B. thuringiensis subsp. konkukian ATP synthase subunit delta (UniProt: Q6HAX6) can be compared with homologs from other species

• Multiple sequence alignment reveals:

  • Highly conserved regions likely involved in critical functions

  • Variable regions potentially associated with species-specific adaptations

  • Conservation patterns related to structural elements (e.g., helices, loops)

Phylogenetic distribution:

• ATP synthase subunit delta is widely distributed across bacterial phyla

• Sequence similarity tends to correlate with taxonomic relationships

• Within the Bacillus genus, higher sequence identity percentages (typically >70%) are observed

Structural conservation:

Functional motifs:

• Key interaction surfaces for binding to other ATP synthase components show higher conservation

• Regions involved in conformational changes during catalysis are typically more conserved than peripheral regions

For researchers interested in evolutionary analyses, focusing on patterns of conservation can provide valuable insights into structure-function relationships and guide experimental designs for mutational studies.

What structural and functional differences exist between ATP synthase components from different bacterial species?

Comparing ATP synthase components across bacterial species reveals important structural and functional adaptations:

Structural variations:

Functional adaptations:

• ATP synthases from extremophiles show adaptations to their environmental conditions:

  • Thermophiles (like Thermotoga maritima mentioned in search result ) have more rigid, thermostable structures

  • Alkaliphiles contain modifications for functioning at high pH

  • Acidophiles have adaptations for operating at low pH

Interaction interfaces:

• The mode of interaction between subunit delta and other components may differ between species

• These differences can affect the efficiency of energy coupling and ATP synthesis

• Comparative analysis can reveal species-specific interaction networks

Regulatory mechanisms:

• Different bacterial species may employ varied regulatory mechanisms for ATP synthase

• Post-translational modifications may differ between species

• Allosteric regulation sites can vary in location and sensitivity

Evolutionary implications:

• Different selection pressures have shaped ATP synthase components

• Horizontal gene transfer events may have contributed to diversity in some lineages

• Co-evolution patterns between interacting subunits can provide insights into functional coupling

For researchers studying B. thuringiensis ATP synthase subunit delta, understanding these differences is crucial for interpreting experimental results and designing functional studies that account for species-specific characteristics.

How can ATP synthase subunit delta be utilized as a target for antimicrobial research?

ATP synthase represents a potential target for antimicrobial development, and understanding subunit delta could contribute to this research area:

Rationale for targeting ATP synthase:

• Essential for bacterial energy metabolism

• Structural differences exist between bacterial and human ATP synthases

• Inhibition would broadly impact bacterial cellular functions

Strategies for targeting subunit delta:

• Structure-based drug design:

  • Use computational models to identify potential binding pockets

  • Perform virtual screening of compound libraries

  • Test high-scoring candidates for inhibitory activity

  • Optimize lead compounds based on structure-activity relationships

• Peptide-based inhibitors:

  • Design peptides that mimic interaction interfaces of subunit delta

  • Test their ability to disrupt complex assembly

  • Optimize peptide stability and cell penetration

  • Evaluate antimicrobial efficacy in vitro and in vivo

• Antibody-based approaches:

  • Generate antibodies against accessible epitopes of subunit delta

  • Test for inhibition of ATP synthase function

  • Evaluate potential for immunotherapy applications

Experimental evaluation methods:

• ATP synthesis inhibition assays using reconstituted systems

• Growth inhibition assays with potential inhibitors

• Membrane potential measurements to assess impact on proton motive force

• Kill kinetics determination for promising compounds

Considerations for antimicrobial development:

• Selectivity for bacterial over mammalian ATP synthase

• Penetration of bacterial cell envelope

• Potential for resistance development

• Spectrum of activity across bacterial species

Research focusing on B. thuringiensis ATP synthase may have broader implications for developing antimicrobials against related pathogenic species within the Bacillus cereus group.

What role might ATP synthase play in B. thuringiensis adaptation to environmental conditions?

ATP synthase function is likely crucial for B. thuringiensis adaptation to various environmental conditions, with potential research implications:

Energy metabolism adaptation:

• B. thuringiensis undergoes significant metabolic changes during different growth phases

• ATP synthase regulation may be integrated with sporulation and toxin production pathways

• Adaptation to fluctuating nutrient availability may involve modulation of ATP synthase activity

Stress response mechanisms:

• Under environmental stresses (pH, temperature, nutrient limitation), energy metabolism must be optimized

• ATP synthase function may be regulated to balance energy production with cellular needs

• Potential research approaches:

  • Monitor ATP synthase gene expression under various stress conditions

  • Assess post-translational modifications in response to stress

  • Measure ATP synthesis capacity during adaptation to different environments

  • Compare ATP synthase activity between vegetative cells and spores

Relationship to virulence factors:

• B. thuringiensis is known for producing delta-endotoxins (Cry proteins)

• Energy demands for toxin production may require coordinated regulation of ATP synthase

• Research has shown that medium components like FeSO4, K2HPO4, starch, and soybean meal influence delta-endotoxin production

• Potential research questions:

  • Is ATP synthase activity coordinated with toxin production?

  • Do energy metabolism pathways influence toxin yield?

  • Can manipulation of ATP synthase affect virulence factor production?

Comparative ecology approach:

• Comparison with related species like B. thuringiensis subsp. israelensis (which produces Cry toxins active against dipteran larvae)

• Assessment of ATP synthase adaptation in strains from different ecological niches

• Evaluation of energy metabolism differences between insecticidal and non-insecticidal strains

What are common challenges in expressing and purifying recombinant ATP synthase components?

Researchers working with ATP synthase components often encounter several challenges that require specific troubleshooting approaches:

Expression challenges:

• Low expression levels

  • Solution: Optimize codon usage for expression host

  • Screen different promoter systems

  • Test various induction conditions (temperature, inducer concentration, timing)

• Protein insolubility

  • Solution: Express as fusion protein with solubility-enhancing tags (MBP, SUMO, TrxA)

  • Lower induction temperature (16-20°C)

  • Add osmolytes or folding enhancers to growth media

  • Consider cell-free expression systems

• Toxicity to expression host

  • Solution: Use tightly regulated expression systems

  • Employ specialized strains designed for toxic protein expression

  • Use lower copy number plasmids

Purification challenges:

• Aggregation during purification

  • Solution: Include stabilizing agents (glycerol, specific salt concentrations)

  • Add mild detergents for membrane-associated components

  • Optimize buffer conditions (pH, ionic strength)

  • Consider on-column refolding protocols

• Co-purification of contaminants

  • Solution: Implement multi-step purification strategy

  • Include additional washing steps with higher salt or low concentrations of denaturants

  • Consider ion exchange chromatography as an orthogonal purification step

• Proteolytic degradation

  • Solution: Add protease inhibitors throughout purification

  • Decrease purification time and temperature

  • Remove flexible regions prone to proteolysis through construct design

Quality control methods:

• SDS-PAGE and western blotting to assess purity and integrity

• Size exclusion chromatography to evaluate oligomeric state

• Mass spectrometry to confirm identity and detect modifications

• Circular dichroism to verify secondary structure content

• Thermal shift assays to assess stability under different buffer conditions

How can researchers verify the functional integrity of purified recombinant ATP synthase subunit delta?

Ensuring that purified recombinant ATP synthase subunit delta retains its functional properties is crucial for meaningful experiments:

Structural integrity assessment:

• Circular dichroism (CD) spectroscopy:

  • Measure far-UV CD spectrum (190-260 nm) to assess secondary structure content

  • Compare with predicted secondary structure based on computational models

  • Perform thermal denaturation to determine stability

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS):

  • Analyze oligomeric state and homogeneity

  • Determine absolute molecular weight

  • Detect potential aggregation

Functional assays:

• Binding assays with interacting partners:

  • Surface plasmon resonance (SPR) with other ATP synthase components

  • Isothermal titration calorimetry (ITC) to measure binding thermodynamics

  • Microscale thermophoresis (MST) for sensitive detection of interactions

• Reconstitution experiments:

  • Combine with other purified ATP synthase components

  • Assess complex formation by native PAGE or analytical ultracentrifugation

  • Measure ATP synthesis/hydrolysis activity of reconstituted complexes

  • Compare activity with and without subunit delta to assess functional contribution

Validation approaches:

• Limited proteolysis to probe folding and domain organization:

  • Incubate with proteases at low concentrations

  • Analyze digestion patterns by SDS-PAGE and mass spectrometry

  • Compare with predictions based on structural models

• Thermal stability assays:

  • Differential scanning fluorimetry (DSF) to measure unfolding transitions

  • Test stability in different buffer conditions

  • Assess effects of potential ligands or interacting partners on stability

By combining multiple complementary approaches, researchers can gain confidence in the structural and functional integrity of their purified recombinant ATP synthase subunit delta before proceeding to more complex experiments.