Recombinant Agrobacterium radiobacter ATP synthase subunit delta (atpH)

What is the structural role of the delta subunit in bacterial F₁F₀-ATP synthase?

The delta (δ) subunit in bacterial ATP synthase serves as a critical component of the peripheral stalk, connecting the F₁ catalytic domain to the membrane-embedded F₀ domain. Based on structural studies of bacterial ATP synthases, the delta subunit functions as a transfer element of elastic energy during ATP formation. In mycobacterial ATP synthases, movements of the peripheral stalk subunit δ have been visualized in different states, underpinning its function in energy transfer during ATP synthesis . The subunit helps maintain proper alignment between the rotating central stalk and the stationary parts of the enzyme, allowing efficient energy coupling between proton translocation and ATP synthesis.

How does the delta subunit of A. radiobacter ATP synthase compare to homologous subunits in other bacteria?

While direct comparative data for A. radiobacter delta subunit is limited in the provided search results, structural studies of other bacterial ATP synthases reveal species-specific adaptations. For instance, mycobacterial ATP synthases contain unique inserted domains in the delta subunit that are not present in other bacteria . These inserted domains may contribute to specialized regulatory mechanisms. When conducting comparative analyses, researchers should focus on:

Sequence alignment and structural prediction tools can help identify conserved regions and species-specific adaptations in the A. radiobacter delta subunit compared to well-characterized homologs.

What expression systems are most effective for producing recombinant ATP synthase subunits?

Multiple expression systems have been successfully utilized for recombinant ATP synthase subunits. Based on experimental evidence, both E. coli and homologous expression systems have proven effective. For recombinant expression of ATP synthase components:

• E. coli expression systems: Allow high-yield production of individual subunits, particularly when codon optimization is applied. This approach was successfully used for expressing F₁-ATPase components from various bacteria .

• Homologous expression: When studying function within the native complex, expressing the recombinant subunit in a deletion strain of the same organism can be advantageous. This approach was demonstrated with the uncB gene (encoding subunit a) in E. coli .

• Eukaryotic systems: For more complex functional studies, mammalian cell lines can be utilized, as demonstrated by the successful expression of ATP5B-paGFP fusion in lung cancer cells to study ATP synthase dynamics .

When selecting an expression system, consider protein folding requirements, post-translational modifications, and how the experimental design will assess functionality.

How can one design experiments to study the specific contribution of the delta subunit to ATP synthase function?

Designing experiments to elucidate the role of the delta subunit requires multiple complementary approaches:

• Genetic deletion and complementation: Create a delta subunit deletion strain and complement with wild-type or mutated versions. Similar approaches have been successful with other ATP synthase subunits, as demonstrated in E. coli where a strain with complete deletion of the chromosomal uncB gene encoding subunit a was constructed . Complementation with modified versions allowed characterization of critical residues in proton translocation.

• Site-directed mutagenesis: Target conserved residues based on sequence alignments to identify functionally important amino acids. This approach revealed that the unique mycobacterial γ-loop and subunit δ are critical elements required for ATP formation .

• Rotary dynamics studies: Employ single-molecule techniques to observe the impact of delta subunit modifications on rotational mechanics. Rotary dynamics studies of recombinant complexes provided insights into chemo-mechanical coupling and regulation mechanisms .

• Cryo-EM structural analysis: Determine structures in different nucleotide-bound states to visualize conformational changes. This approach successfully visualized critical elements for latent ATP hydrolysis and efficient ATP synthesis in mycobacterial ATP synthases .

• ATP synthesis/hydrolysis assays: Measure enzymatic activity with reconstituted complexes containing wildtype or modified delta subunits to quantify functional impacts.

What methods are most effective for purifying functional recombinant ATP synthase subunits?

Purification of functional ATP synthase subunits requires careful consideration of protein stability and maintaining native conformation:

• Affinity chromatography: Utilize His-tagged constructs for initial capture, but be mindful of tag position to avoid functional interference. N-terminal or C-terminal placement should be determined based on structural information to prevent disruption of interaction surfaces.

• Detergent selection: For membrane-associated components, detergent screening is crucial. Mild detergents like DDM (n-dodecyl β-D-maltoside) or digitonin typically preserve protein-protein interactions within the complex.

• Two-step purification strategy:

  • First step: Affinity chromatography (e.g., IMAC for His-tagged proteins)

  • Second step: Size exclusion chromatography to isolate properly folded proteins and remove aggregates

• Functional verification: Assess ATP hydrolysis activity using colorimetric assays (e.g., malachite green phosphate detection) to confirm that purified components retain functionality.

• Complex reconstitution: For studying delta subunit in context, reconstitution experiments combining recombinant subunits with partially assembled complexes can be effective, as demonstrated in studies with recombinant mycobacterial F₁-ATPase .

How do mutations in the delta subunit affect ATP synthase assembly and function?

Mutations in the delta subunit can have profound effects on both assembly and function of the ATP synthase complex:

Research with mycobacterial ATP synthase demonstrated that mutational studies of components like subunit δ can reveal their critical importance for ATP formation . When characterizing delta subunit mutations, researchers should assess:

• Assembly state via blue native PAGE or co-immunoprecipitation with other subunits

• ATP synthesis activity in reconstituted systems

• ATP hydrolysis regulation

• Growth phenotypes in complementation strains

• Structural integrity via limited proteolysis or thermal stability assays

What are the challenges in expressing and analyzing ATP synthase subunits in heterologous systems?

Heterologous expression of ATP synthase components presents several challenges:

• Protein misfolding: The structural complexity of ATP synthase subunits often leads to misfolding in heterologous systems. Using molecular chaperones as co-expression partners can improve folding outcomes.

• Membrane insertion: For components with transmembrane domains, proper insertion into membranes may require specific machinery. Studies with E. coli demonstrated that while subunit a is not required for the insertion of subunits b and c , other assembly factors may be needed.

• Post-translational modifications: Species-specific modifications may be absent in heterologous systems, affecting function or stability.

• Assessing functionality: Individual subunits may not display measurable activity outside the intact complex. Researchers should design partial assembly reconstitution experiments to test functionality, as demonstrated in studies with recombinant F₁-ATPase .

• Species-specific interactions: The unique structural elements of ATP synthases from different species may not be accommodated in heterologous complexes. For example, mycobacterial ATP synthases contain specific structural elements (γ-loop, inserted δ-domain) that play critical roles in function .

To overcome these challenges, researchers can employ co-expression of interacting partners, optimize expression conditions (temperature, induction time), and use solubility tags designed for membrane proteins.

What advanced imaging techniques are most informative for studying ATP synthase delta subunit structure?

Several advanced imaging techniques provide valuable insights into ATP synthase subunit structure:

• Cryo-electron microscopy (cryo-EM): Currently the gold standard for ATP synthase structural studies, providing near-atomic resolution of the entire complex. Recent studies utilized cryo-EM to visualize ATP synthase structures with different nucleotide occupations within catalytic sites . This approach can reveal critical structural elements and conformational changes during the catalytic cycle.

• X-ray crystallography: While challenging for the entire complex, this technique can provide high-resolution structures of individual domains or subunits, including the delta subunit.

• Single-molecule FRET: For dynamics studies, fluorescently labeled delta subunits can reveal conformational changes during rotation. Similar approaches using paGFP fusion proteins have been used to study ATP synthase dynamics .

• NMR spectroscopy: For smaller domains or flexible regions, solution NMR can provide structural and dynamic information at the atomic level.

• Cross-linking mass spectrometry: Identifies interaction interfaces between delta and other subunits by chemically cross-linking proximal residues and analyzing via mass spectrometry.

When applying these techniques to A. radiobacter delta subunit studies, researchers should consider stabilizing the complex in specific conformational states using nucleotide analogs, inhibitors, or engineered disulfide bonds.

How does the delta subunit contribute to the regulation of ATP synthase activity?

The delta subunit plays multiple regulatory roles in ATP synthase function, acting as both a structural element and potential regulatory target:

• Coupling efficiency: As part of the peripheral stalk, delta subunit influences the elastic coupling between F₁ and F₀ domains. Studies have shown that the peripheral stalk subunit δ functions as a transfer element of elastic energy during ATP formation .

• Rotational dynamics: The delta subunit may influence the transition between active and inhibited states. In mycobacterial ATP synthase, rotational studies indicated that the transition between inhibition states is a rapid process, with specific domains playing regulatory roles .

• Species-specific regulation: Unique structural elements in the delta subunit can confer species-specific regulatory mechanisms. Mycobacterial ATP synthases contain unique elements that are critical for function and represent potential targets for species-specific inhibitors .

To study these regulatory functions, researchers should combine structural approaches with functional assays measuring ATP synthesis/hydrolysis under various conditions (pH, membrane potential, nucleotide concentrations) while introducing specific mutations to the delta subunit.

What insights can molecular dynamics simulations provide about delta subunit function?

Molecular dynamics (MD) simulations offer valuable insights into dynamic aspects of delta subunit function that may be difficult to capture experimentally:

• Conformational flexibility: MD simulations can reveal flexible regions and conformational changes during the catalytic cycle, particularly how the delta subunit responds to rotation of the central stalk.

• Energy transfer mechanisms: Simulations can elucidate how mechanical energy is transferred through the peripheral stalk during rotational catalysis. This is particularly relevant as the delta subunit has been implicated as a transfer element of elastic energy during ATP formation .

• Water dynamics and proton pathways: For subunits involved in proton translocation, MD simulations can reveal water-mediated proton transfer pathways and conformational changes that gate proton movement.

• Interaction energetics: Binding free energy calculations can quantify interaction strengths between delta and other subunits under different conformational states.

• Effects of mutations: In silico mutations can predict functional consequences before experimental validation, helping to prioritize mutations for laboratory testing.

When conducting MD simulations, researchers should ensure proper parameterization of the protein-membrane system and consider enhanced sampling techniques to capture rare conformational transitions relevant to the catalytic cycle.

How can researchers overcome challenges in expressing insoluble ATP synthase subunits?

Expression of soluble, functional ATP synthase subunits often presents challenges due to their hydrophobic nature and complex folding requirements:

• Fusion tags for solubility enhancement:

  • MBP (maltose-binding protein) tag: Highly soluble partner that can improve folding

  • SUMO tag: Enhances solubility and can be precisely removed with SUMO protease

  • Thioredoxin: Promotes disulfide bond formation in the cytoplasm

• Expression conditions optimization:

  • Lower temperature (16-25°C): Slows protein synthesis, allowing more time for proper folding

  • Reduced inducer concentration: Prevents overwhelming cellular folding machinery

  • Co-expression with chaperones: GroEL/ES, DnaK/J systems can assist folding

• Solubilization strategies:

  • Screen multiple detergents: Start with mild detergents (DDM, LMNG, digitonin)

  • Detergent concentration gradient: Optimize minimal concentration needed for solubilization

  • Lipid supplementation: Adding specific lipids can stabilize native conformation

• Refolding protocols:

  • Inclusion body isolation followed by controlled refolding

  • Step-wise dialysis to gradually remove denaturants

  • On-column refolding during affinity purification

For the delta subunit specifically, identification of stable domains through bioinformatic analysis may allow expression of functional subdomains if the full-length protein proves recalcitrant to soluble expression.

What are the best methods for analyzing protein-protein interactions involving the delta subunit?

Multiple complementary techniques can effectively analyze interactions between the delta subunit and other components of the ATP synthase complex:

• Co-immunoprecipitation (Co-IP): Using antibodies against the delta subunit or potential interacting partners to pull down complexes from cell lysates. This approach can identify physiologically relevant interactions.

• Yeast two-hybrid (Y2H): While limited to soluble domains, Y2H can identify direct binary interactions and map interaction sites through domain analysis.

• Surface plasmon resonance (SPR): Provides quantitative binding kinetics and affinity measurements between purified components, allowing comparison between wildtype and mutant variants.

• Cross-linking coupled with mass spectrometry: Chemical cross-linkers covalently link proximal residues, and subsequent MS analysis identifies interaction interfaces at amino acid resolution.

• Fluorescence techniques:

  • FRET (Förster Resonance Energy Transfer): Measures proximity between fluorescently labeled proteins

  • FCCS (Fluorescence Cross-Correlation Spectroscopy): Detects co-diffusion of labeled proteins in solution

• Structure-based methods:

  • Hydrogen-deuterium exchange MS: Maps interaction interfaces through differential solvent accessibility

  • Cryo-EM: Directly visualizes interaction interfaces in the assembled complex

When designing interaction studies, researchers should consider the native membrane environment for transmembrane components, as detergent solubilization may disrupt some interactions.

How can isotopic labeling enhance structural studies of ATP synthase components?

Isotopic labeling provides powerful tools for structural analysis of ATP synthase components, particularly when conventional approaches are challenging:

• NMR spectroscopy applications:

  • ¹⁵N/¹³C labeling: Enables backbone and side-chain assignments for structural determination

  • Selective amino acid labeling: Simplifies spectra for large proteins like the delta subunit

  • Methyl-group labeling: Provides probes for studying dynamics in large protein complexes

  • TROSY techniques with deuteration: Improves spectral quality for high molecular weight complexes

• Mass spectrometry applications:

  • ¹⁸O labeling: Distinguishes newly synthesized from existing proteins in turnover studies

  • Hydrogen-deuterium exchange: Maps solvent accessibility and conformational changes

  • SILAC labeling: Quantifies protein-protein interactions and complex formation

• Neutron scattering:

  • Deuteration: Provides contrast for neutron scattering experiments to locate specific domains within the complex

  • Contrast matching: Selectively highlights specific subunits within the assembled complex

• EPR spectroscopy:

  • Site-directed spin labeling: Measures distances between specific residues

  • DEER/PELDOR: Determines long-range distances between paramagnetic centers

These techniques can reveal structural dynamics not captured by static structural methods and are particularly valuable for understanding the conformational changes of the delta subunit during the catalytic cycle of ATP synthase.

What controls should be included when analyzing the function of recombinant ATP synthase delta subunit?

Rigorous controls are essential when analyzing recombinant ATP synthase delta subunit function:

• Genetic complementation controls:

  • Negative control: Delta subunit deletion strain showing loss of function

  • Positive control: Wildtype delta subunit complementation restoring function

  • Empty vector control: Ruling out vector-related effects

• Protein quality controls:

  • Size exclusion chromatography: Confirms proper oligomeric state

  • Circular dichroism: Verifies secondary structure content

  • Thermal shift assays: Assesses protein stability

  • Limited proteolysis: Tests for proper folding

• Functional assay controls:

  • Known inhibitors: Oligomycin for FₒF₁ complexes validates assay specificity

  • Uncouplers: FCCP/CCCP confirm proton gradient dependency

  • ATP hydrolysis: Verifies catalytic function in reverse direction

  • Known mutations: Previously characterized mutations serve as benchmarks

• Interaction studies controls:

  • Non-interacting protein: Controls for non-specific binding

  • Competition assays: Validate specificity of observed interactions

  • Truncated constructs: Map minimal interaction domains

These controls are essential for distinguishing specific effects from artifacts, particularly when working with complex multi-subunit assemblies like ATP synthase. Similar approaches were used in studies of E. coli ATP synthase, where functional complementation of deletion strains helped characterize critical residues in the a subunit .

How might structural differences in the A. radiobacter delta subunit be exploited for antimicrobial development?

Species-specific structural elements in bacterial ATP synthases offer promising targets for selective antimicrobial development:

• Exploitable structural differences:

  • Unique binding pockets or interfaces specific to A. radiobacter

  • Regulatory mechanisms not present in human ATP synthases

  • Species-specific insertion domains or loops

Research on mycobacterial ATP synthases has demonstrated that species-specific elements like the γ-loop, inserted δ-domain, and C-terminal domain (αCTD) of subunit α represent attractive targets for developing species-specific inhibitors . Similar approaches could be applied to A. radiobacter delta subunit.

• Target-based strategies:

  • Structure-based virtual screening against identified binding sites

  • Fragment-based drug discovery focusing on species-specific pockets

  • Peptidomimetics targeting unique protein-protein interfaces

• Validation approaches:

  • Biochemical assays measuring ATP synthesis inhibition

  • Growth inhibition assays in bacterial cultures

  • Resistance development monitoring

  • Selectivity profiling against human ATP synthase

• Combination approaches:

  • Dual targeting of ATP synthase and other essential processes

  • Synergistic inhibitor pairs targeting different ATP synthase subunits

These approaches could lead to narrow-spectrum antimicrobials with reduced resistance development and fewer side effects compared to broad-spectrum agents.

What insights can comparative studies of ATP synthase delta subunits across bacterial species provide?

Comparative analysis of ATP synthase delta subunits across bacterial species offers valuable insights into evolutionary adaptation and functional specialization:

• Evolutionary conservation patterns:

  • Core functional domains: Identifying universally conserved regions essential for ATP synthase function

  • Species-specific adaptations: Regions that diverge in response to environmental niches

  • Co-evolution analysis: Identifying coordinated changes between interacting subunits

• Structural adaptations:

  • Membrane composition adaptations: Modifications for function in different lipid environments

  • Thermostability mechanisms: Structural features in thermophilic vs. mesophilic bacteria

  • pH adaptations: Modifications for function at acidic/alkaline conditions

• Regulatory mechanisms:

  • Diverse regulatory elements: Species-specific regulation of ATP synthesis/hydrolysis

  • Environmental response elements: Adaptations to energy status or stress conditions

• Methodological approaches:

  • Phylogenetic analysis: Evolutionary relationships between delta subunit variants

  • Structural comparisons: Homology modeling based on known structures

  • Functional complementation: Cross-species complementation testing

Mycobacterial ATP synthases demonstrate species-specific structural elements that contribute to their unique functional properties . Similar comparative approaches with A. radiobacter delta subunit could reveal adaptations specific to plant-associated bacteria.

What are the most promising future research directions for ATP synthase delta subunit studies?

Several promising research directions emerge for ATP synthase delta subunit investigations:

• Structural dynamics during catalysis:

  • Time-resolved cryo-EM to capture transient states

  • Single-molecule studies monitoring conformational changes during rotation

  • Computational simulations of energy transfer through the peripheral stalk

• Species-specific therapeutic targeting:

  • Structure-based design of selective inhibitors targeting unique features

  • Allosteric modulators affecting delta subunit interactions

  • Peptide inhibitors disrupting critical protein-protein interfaces

• Synthetic biology applications:

  • Engineering modified ATP synthases with altered regulatory properties

  • Building minimal ATP synthase systems with redesigned delta subunits

  • Creating hybrid systems with components from different species

• Fundamental bioenergetic questions:

  • Elucidating the precise mechanism of energy transfer through the peripheral stalk

  • Understanding species-specific adaptations to different environmental conditions

  • Mapping the co-evolution of interacting surfaces between subunits

• Methodological advances:

  • Developing improved expression systems for membrane protein complexes

  • Applying integrative structural biology approaches combining multiple techniques

  • Establishing high-throughput functional assays for ATP synthase activity

These directions will benefit from interdisciplinary approaches combining structural biology, biochemistry, computational modeling, and synthetic biology to fully understand this fascinating molecular machine.

How does research on bacterial ATP synthase contribute to our understanding of bioenergetic systems?

Research on bacterial ATP synthase, including the delta subunit, provides fundamental insights into bioenergetic systems across all domains of life:

• Evolutionary perspectives:

  • ATP synthase represents an ancient molecular machine conserved across bacteria, archaea, and eukaryotes

  • Bacterial systems often serve as simpler models for understanding more complex eukaryotic counterparts

  • Comparative studies reveal both conserved principles and diverse adaptations

• Mechanistic understanding:

  • Rotary catalysis principles established in bacterial systems apply broadly

  • Energy coupling mechanisms between proton translocation and ATP synthesis illuminate fundamental bioenergetic principles

  • Regulatory mechanisms reveal diverse strategies for energy conservation

• Medical relevance:

  • Bacterial ATP synthases represent targets for antimicrobial development

  • Understanding bacterial systems informs mitochondrial disease mechanisms

  • Species-specific elements, as identified in mycobacterial ATP synthases , can be exploited for selective targeting

• Biotechnological applications:

  • Bacterial ATP synthases can be engineered for bioenergy applications

  • Understanding energy efficiency principles can inspire biomimetic energy systems

  • Bacterial components can be incorporated into hybrid systems with novel properties

• Fundamental physics:

  • ATP synthase exemplifies biological nanomachines operating near thermodynamic efficiency

  • Studying energy transduction clarifies principles of molecular motors

  • Conformational coupling illustrates information transfer in macromolecular assemblies

These contributions highlight the centrality of ATP synthase research to our understanding of life's essential energy conversion systems.

What methodological advances would most benefit the study of ATP synthase subunits?

Several methodological advances would significantly enhance ATP synthase subunit research:

• Structural biology improvements:

  • Higher resolution cryo-EM for membrane proteins in lipid environments

  • Time-resolved structural methods capturing transient states

  • Improved computational methods for modeling dynamic assemblies

  • Enhanced mass spectrometry approaches for membrane protein complexes

• Expression and purification advances:

  • Improved membrane protein expression systems with higher yields

  • Novel detergents or nanodiscs better mimicking native environments

  • High-throughput purification strategies for mutant screening

  • Cell-free expression systems for toxic membrane proteins

• Functional assay developments:

  • Single-molecule techniques with improved spatial and temporal resolution

  • High-throughput ATP synthesis/hydrolysis assays compatible with crude preparations

  • Improved proton translocation measurements with higher sensitivity

  • In vivo assays correlating structure with physiological function

• Computational method enhancements:

  • More accurate force fields for membrane protein simulations

  • Enhanced sampling methods for capturing rare conformational transitions

  • Improved protein-protein docking accounting for flexibility

  • Integration of experimental constraints with computational models

• Genetic tool developments:

  • CRISPR-based methods for precise genomic editing in diverse bacterial species

  • Regulated expression systems for essential proteins like ATP synthase components

  • Improved selection methods for functional complementation

  • Site-specific incorporation of non-canonical amino acids for probing function