Recombinant Salmonella paratyphi C ATP synthase subunit c (atpE)

What is the structural composition of Salmonella paratyphi C ATP synthase subunit c (atpE)?

Salmonella paratyphi C ATP synthase subunit c (atpE) is a relatively small protein consisting of 79 amino acids with the following sequence: MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . This highly conserved protein forms part of the F0 sector of ATP synthase, specifically contributing to the formation of the c-ring structure. The protein contains hydrophobic regions that facilitate its integration into membranes, which is essential for its role in proton translocation across the bacterial membrane. When produced as a recombinant protein, it is typically fused with an N-terminal His-tag to facilitate purification and experimental manipulation .

How does recombinant S. paratyphi C atpE compare with other Salmonella species?

Comparative analysis of ATP synthase subunit c across different Salmonella species reveals remarkable conservation. The amino acid sequence of Salmonella paratyphi C atpE (UniProt ID: C0Q2N7) is identical to that of Salmonella arizonae atpE (UniProt ID: A9MJR4) . This perfect sequence homology suggests:

• Evolutionary conservation of this critical component

• Functional importance across Salmonella species

• Potential for cross-species experimental applications

This conservation allows researchers to apply findings from one Salmonella species to others, though regulatory elements and interaction partners may still differ between species. Despite identical sequences, researchers should note that species-specific post-translational modifications might still differentiate the functional properties of these proteins in their native bacterial contexts.

What experimental approaches are suitable for expressing and purifying recombinant S. paratyphi C atpE?

The expression and purification of recombinant S. paratyphi C atpE typically employs the following methodological approach:

Expression System:

• E. coli-based expression systems are the preferred host for recombinant production

• Expression vectors incorporating N-terminal His-tags facilitate downstream purification

• Induction conditions require optimization for membrane protein expression

Purification Protocol:

• Bacterial cell lysis under conditions that preserve membrane protein structure

• Affinity chromatography using nickel or cobalt resins to capture His-tagged proteins

• Further purification via size exclusion or ion exchange chromatography if necessary

• Final preparation as a lyophilized powder for storage stability

Storage Considerations:

• Store at -20°C/-80°C in aliquots to prevent freeze-thaw cycles

• Working aliquots should be maintained at 4°C for no more than one week

• Reconstitution in deionized water to 0.1-1.0 mg/mL, with addition of 5-50% glycerol for long-term storage

These methodological approaches ensure >90% purity as confirmed by SDS-PAGE analysis, suitable for downstream functional and structural studies.

How does atpE contribute to ATP synthase function in Salmonella?

The c subunit (atpE) plays a critical role in the rotary mechanism of ATP synthase, primarily by facilitating proton translocation across the membrane. In the functional ATP synthase complex:

• Multiple c subunits assemble into a ring structure within the membrane

• Proton translocation through the F0 sector drives rotation of this c-ring relative to the a-subunit

• This rotational energy is transferred to the F1 sector, powering ATP synthesis

• Key glutamic acid residues in different c-subunits contribute to proton release to and uptake from the a-subunit

The proton motive force generated across the bacterial membrane energizes this rotational mechanism. Experimental evidence indicates that mutations in the c subunit can significantly impact ATP synthesis and proton pump activities, demonstrating its essential role in energy transduction . The c-ring's structure and function are highly conserved across bacterial species, suggesting evolutionary importance of this mechanism for cellular bioenergetics.

What methodologies can be used to study the proton translocation function of recombinant atpE?

Researchers investigating the proton translocation function of recombinant atpE can employ several sophisticated methodological approaches:

Liposome Reconstitution Systems:

• Co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes

• Creation of functionally coupled enzyme systems via proton translocation

• Measurement of ATP synthesis rates under steady-state conditions (up to 90 ATP×s⁻¹×enzyme⁻¹)

• Introduction of ionophores (e.g., FCCP, SF6847) at controlled concentrations to study uncoupling effects

Mutation Analysis:

• Site-directed mutagenesis of key residues (particularly glutamic acid positions)

• Comparison of wild-type and mutant proteins (e.g., E56D mutations) to assess functional impact

• Analysis of single versus multiple mutations to detect cooperative effects between c-subunits

Advanced Biophysical Techniques:

• Fluorescence-based assays to monitor proton gradient formation

• Membrane potential measurements using potentiometric dyes

• Real-time ATP synthesis monitoring using luciferase-based detection systems

These methodologies provide complementary approaches to understanding the complex bioenergetic processes mediated by the atpE protein within the ATP synthase complex.

What role does cooperation among c-subunits play in ATP synthase function, and how can this be experimentally investigated?

Recent research has uncovered significant cooperation among c-subunits in ATP synthase function that can be examined through several experimental approaches:

Evidence of Cooperation:

• ATP synthesis and proton pump activities decrease with single c-subunit mutations

• Further decreases occur with double mutations, demonstrating functional coupling

• Activity decreases as the distance between mutation sites increases, indicating spatial cooperation between c-subunits

Experimental Investigation Methods:

• Genetic Engineering Approach:

  • Creation of genetically fused single-chain c-rings

  • Introduction of specific mutations (e.g., E56D) at defined positions

  • Analysis of ATP synthesis capacity with various mutation combinations and positions

• Computational Simulation:

  • Proton transfer-coupled molecular dynamics simulations

  • Analysis of simulation trajectories to determine proton uptake duration times

  • Correlation of computational findings with biochemical assay results

• Functional Assays:

  • Measurement of ATP synthesis rates under controlled conditions

  • Proton pumping assays with pH-sensitive fluorescent indicators

  • Comparison of wild-type and mutant enzyme kinetics

The experimental evidence reveals that prolonged proton uptake times in mutated c-subunits can be shared between subunits, with the degree of time-sharing decreasing as the distance between mutation sites increases . This mechanism explains the observed cooperation in biochemical assays and provides insight into the rotational dynamics of the ATP synthase complex.

How does ATP synthase function relate to Salmonella virulence mechanisms?

The relationship between ATP synthase function and Salmonella virulence involves sophisticated regulatory mechanisms:

MgtC Virulence Factor Interaction:

• The MgtC virulence protein, required for intraphagosomal replication, directly interacts with and inhibits the F1F0 ATP synthase

• This interaction reduces ATP levels within the bacterium

• By lowering ATP levels, MgtC prevents a rise in cyclic diguanylate (c-di-GMP), a second messenger that promotes biofilm formation

Metabolic Regulation and Virulence:

• ATP synthase activity influences intracellular ATP concentrations

• ATP levels affect expression of virulence-associated genes, including cellulose biosynthesis

• Inactivation of MgtC results in increased bcsA mRNA (sevenfold increase), indicating deregulation of cellulose synthase expression

Experimental Evidence:
The expression of α, β, and γ components of the F1 subunit of ATP synthase prevents cellulose production in MgtC mutants, confirming that ATP accumulation drives the phenotype . This indicates that virulence factors like MgtC function partly by repressing traits (such as cellulose production) that would otherwise interfere with pathogenesis.

What experimental approaches can be used to study ATP synthase inhibition in the context of Salmonella pathogenesis?

Researchers investigating the connection between ATP synthase inhibition and Salmonella pathogenesis can employ multiple methodological approaches:

Cell Culture Models:

• Macrophage infection assays with wild-type and ATP synthase mutant Salmonella

• Measurement of intracellular bacterial replication rates

• Assessment of phagosomal pH regulation in infected cells

• Analysis of cellulose production within the intracellular environment

Genetic Manipulation Approaches:

• Creation of targeted mutations in ATP synthase components

• Development of regulated expression systems for ATP synthase genes

• Complementation studies with wild-type and mutant ATP synthase components

• Dual manipulation of MgtC and ATP synthase to analyze interaction effects

Biochemical Assays:

• Measurement of intracellular ATP levels in various genetic backgrounds

• Quantification of cyclic diguanylate (c-di-GMP) concentrations

• RNA analysis to assess expression of ATP synthase and cellulose synthase genes

• Cellulose detection assays (e.g., calcofluor binding) to correlate with ATP levels

These approaches enable researchers to dissect the complex relationship between bacterial bioenergetics and virulence mechanisms, potentially identifying novel targets for antimicrobial development.

How can synthetic biology approaches be used to investigate c-subunit function in Salmonella ATP synthase?

Synthetic biology offers powerful tools for investigating c-subunit function in Salmonella ATP synthase:

Engineered c-ring Constructs:

• Genetically fused single-chain c-rings that enable precise control over the composition of the c-ring

• Introduction of specific mutations at defined positions within the c-ring sequence

• Creation of chimeric c-rings incorporating subunits from different species

• Development of tagged c-subunits for visualization or affinity purification

Functional Reconstitution Systems:

• Co-reconstitution of synthetic c-rings with other ATP synthase components

• Creation of minimal functional systems to isolate c-subunit contributions

• Development of artificial membrane systems with controlled lipid composition

• Integration of ATP synthase complexes with other respiratory chain components

Advantages of Synthetic Approaches:

• Precise control over protein composition and stoichiometry

• Ability to introduce non-natural amino acids at specific positions

• Creation of proteins with novel functional properties

• Systematic investigation of structure-function relationships

These synthetic biology approaches overcome limitations of traditional genetic methods, allowing researchers to address fundamental questions about c-subunit function and cooperation that would be difficult to investigate using conventional techniques.

What are the key considerations for designing molecular dynamics simulations to study atpE function?

Designing effective molecular dynamics (MD) simulations to study atpE function requires careful consideration of multiple factors:

Simulation System Setup:

• Construction of accurate c-ring models based on structural data

• Proper embedding in lipid bilayers that mimic bacterial membranes

• Inclusion of sufficient water molecules and ions to represent physiological conditions

• Consideration of the entire F0 sector versus isolated c-ring simulations

Critical Parameters:

• Force field selection appropriate for membrane protein simulations

• Simulation timescales sufficient to capture relevant proton translocation events

• Temperature and pressure controls to maintain physiological conditions

• Treatment of long-range electrostatic interactions

Advanced Simulation Approaches:

• Proton transfer-coupled MD simulations to model protonation/deprotonation events

• Free energy calculations to quantify energetic barriers to proton transfer

• Enhanced sampling techniques to access rare conformational states

• Quantum mechanics/molecular mechanics (QM/MM) methods for accurate proton transfer modeling

Validation Strategies:

• Comparison with experimental mutation effects

• Verification against known functional properties of the system

• Consistency checks across multiple simulation replicates

• Prediction of novel properties that can be experimentally tested

Properly designed MD simulations can provide atomic-level insights into mechanisms that are difficult to observe experimentally, such as the prolonged duration times for proton uptake observed in mutated c-subunits and how these effects can be shared between subunits .

How conserved is the atpE sequence across different Salmonella species and what are the functional implications?

Analysis of atpE conservation across Salmonella species reveals important evolutionary and functional insights:

Sequence Conservation:

• The 79-amino acid sequence is perfectly conserved between Salmonella paratyphi C and Salmonella arizonae

• This high conservation extends to other Salmonella species and closely related enterobacteria

• Key functional residues, particularly the essential glutamic acid involved in proton translocation, show nearly universal conservation

Conservation Table:

Functional Implications:

• Essential nature of the c-subunit structure and function across species

• Strong evolutionary pressure to maintain specific sequence elements

• Potential for cross-species functional complementation in experimental settings

• Likely conservation of regulatory mechanisms controlling atpE expression

This exceptional sequence conservation supports the use of findings from model organisms to understand atpE function across Salmonella species and suggests that therapeutic approaches targeting atpE would likely have broad efficacy across this bacterial genus.

What experimental approaches can be used to compare functional differences between atpE from various bacterial species?

Comparative functional analysis of atpE from different bacterial species can be accomplished through several methodological approaches:

Heterologous Expression and Complementation:

• Expression of atpE variants from different species in a common host organism

• Complementation studies in atpE knockout strains to assess functional equivalence

• Growth rate and ATP production measurements under various stress conditions

• Competition assays between strains expressing different atpE variants

Biochemical Characterization:

• Purification of recombinant atpE proteins from multiple species

• Comparative analysis of stability, oligomerization, and membrane integration

• Proton binding and translocation assays under controlled conditions

• Structural studies using X-ray crystallography or cryo-electron microscopy

Chimeric Protein Analysis:

• Creation of chimeric c-subunits with domains from different species

• Identification of regions responsible for species-specific functional differences

• Analysis of hybrid ATP synthase complexes with mixed subunit compositions

• Correlation of functional differences with environmental adaptations

Liposome Reconstitution:

• Co-reconstitution of terminal oxidases and ATP synthases from different species

• Measurement of ATP synthesis rates under standardized conditions

• Determination of proton translocation efficiency and coupling ratios

• Assessment of functional responses to temperature, pH, and ionic conditions

These approaches provide a comprehensive framework for understanding how evolutionary divergence affects atpE function across bacterial species, potentially revealing adaptations to specific environmental niches.

What are the emerging research questions regarding S. paratyphi C atpE that remain to be addressed?

Despite significant advances in understanding S. paratyphi C atpE, several critical research questions remain unexplored:

• Regulatory Mechanisms: How is atpE expression regulated in response to environmental conditions relevant to Salmonella pathogenesis?

• Post-translational Modifications: Do specific post-translational modifications of atpE occur during infection, and how do they affect function?

• Host Interactions: Does atpE or the ATP synthase complex interact with host factors during infection?

• Drug Target Potential: Can the unique features of bacterial atpE be exploited for development of selective antimicrobial agents?

• Alternative Functions: Does atpE have moonlighting functions beyond its role in ATP synthesis?

Addressing these questions will require innovative experimental approaches combining structural biology, advanced imaging, genetic manipulation, and infection models. The continued development of synthetic biology tools will be particularly valuable for dissecting the complex relationship between atpE structure, function, and bacterial pathogenesis.

What methodological advances would enhance the study of c-subunit cooperation in ATP synthase function?

Future methodological advances that would significantly enhance our understanding of c-subunit cooperation include:

Technical Innovations:

• Single-molecule imaging techniques to visualize c-ring rotation in real time

• High-resolution cryo-electron microscopy to capture different conformational states during rotation

• Time-resolved structural methods to track proton movement through the complex

• Advanced computational approaches for simulating complete rotational cycles

Experimental Systems:

• Expanded genetic toolkits for precise manipulation of c-subunit stoichiometry and composition

• Microfluidic platforms for high-throughput analysis of ATP synthase variants

• In vitro systems that mimic the natural membrane environment more accurately

• Methods for studying ATP synthase function in the context of the complete respiratory chain

Integration of Approaches: