Recombinant Shewanella baltica ATP synthase subunit c (atpE)

What is the basic structure and function of ATP synthase in Shewanella baltica?

ATP synthase in Shewanella baltica consists of two major sectors: F₀ (membrane-embedded) and F₁ (catalytic). The F₀ sector contains subunits a, b, and c, with the c-subunit (atpE) forming an oligomeric ring that facilitates proton translocation across the membrane. The F₁ sector contains α, β, γ, δ, and ε subunits, with the catalytic hexamer of alternating α and β subunits responsible for ATP synthesis.

The enzyme catalyzes ATP synthesis from ADP and inorganic phosphate using the energy from proton or sodium ion gradients. In Shewanella baltica, this enzyme is particularly important as it functions efficiently under both aerobic and anaerobic conditions, supporting the organism's versatility as a psychrotrophic bacterium in marine environments.

How does the c-subunit (atpE) specifically contribute to ATP synthase function?

The c-subunit (atpE) forms the rotor ring in the F₀ sector of ATP synthase. Each c-subunit contains a critical proton-binding site, typically an acidic residue (aspartate or glutamate), which undergoes protonation and deprotonation during catalysis. As protons pass through the a/c interface, they cause rotation of the c-ring, which is mechanically coupled to the central stalk (primarily the γ subunit). This rotation drives conformational changes in the F₁ catalytic sites, promoting ATP synthesis.

In Shewanella baltica, the c-subunit is adapted to function across varying environmental conditions, particularly in response to temperature and salinity fluctuations common in marine habitats.

What evolutionary adaptations are observed in Shewanella baltica ATP synthase compared to mesophilic bacteria?

Shewanella baltica, as a psychrotrophic organism, exhibits ATP synthase adaptations for function at lower temperatures. These include:

• Modified amino acid composition in transmembrane regions of the c-subunit, with increased flexibility-conferring residues

• Altered ion-binding sites that maintain efficiency at lower temperatures

• Enhanced stability of subunit interfaces under varied osmotic conditions

Proteomic analyses show that S. baltica specifically adjusts ATP synthase expression in response to salinity changes, optimizing energy production in marine environments. This adaptation involves DNA-binding proteins and polyamine uptake to stabilize nucleoid structure.

What is the recommended expression system for recombinant Shewanella baltica ATP synthase subunit c (atpE)?

For recombinant production of Shewanella baltica ATP synthase subunit c (atpE), heterologous expression in E. coli is the most widely used approach. The methodology typically involves:

• Cloning the atpE gene into an expression vector with a His-tag for purification

• Transformation into an E. coli expression strain (commonly BL21(DE3) or derivatives)

• Induction with IPTG at lower temperatures (16-25°C) to enhance proper folding of membrane proteins

• Expression in minimal media supplemented with appropriate carbon sources

Bacterial fermentation in E. coli, followed by affinity chromatography using a nickel- or cobalt-chelated column is the standard approach due to the incorporated His-tag. Yields can be optimized by adjusting induction parameters and growth conditions to account for the membrane-associated nature of the protein.

What purification strategy yields the highest purity and activity for recombinant atpE?

A multistep purification approach yields the highest purity and retained activity:

• Initial membrane isolation via differential centrifugation

• Detergent solubilization (commonly n-dodecyl β-D-maltoside or digitonin)

• Affinity chromatography using nickel- or cobalt-chelated columns

• Size exclusion chromatography to remove aggregates and contaminating proteins

• Optional ion exchange chromatography for enhanced purity

Post-purification, the protein is typically lyophilized and reconstituted in sterile water with glycerol for stability. Quality assurance must include SDS-PAGE verification and mass spectrometry analysis to confirm molecular weight and sequence integrity.

The table below summarizes key purification parameters:

How can researchers assess the proper folding and oligomeric state of purified atpE?

Multiple complementary techniques should be employed to assess the proper folding and oligomeric state:

• Circular Dichroism (CD) spectroscopy to evaluate secondary structure elements

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) to determine the oligomeric state in detergent micelles

• Blue Native PAGE to analyze the intact c-ring assembly

• Limited proteolysis to assess the accessibility of cleavage sites as indicators of correct folding

• Reconstitution into liposomes followed by proton transport assays to confirm functionality

Each technique provides different insights into protein quality. For instance, CD spectroscopy can confirm the high α-helical content expected for c-subunits, while SEC-MALS can verify the formation of the c-ring oligomer, typically comprising 10-15 subunits depending on the species .

What methods are most suitable for measuring ATP synthase activity in reconstituted systems using Shewanella baltica atpE?

For functional characterization of reconstituted systems containing Shewanella baltica atpE, researchers should consider these methodologies:

• Proton Pumping Assays: Using pH-sensitive fluorescent dyes (ACMA or pyranine) to monitor proton translocation across liposomal membranes

• ATP Synthesis Measurements: Coupling ATP production to NADP+ reduction via hexokinase and glucose-6-phosphate dehydrogenase, measured spectrophotometrically

• Patch-Clamp Electrophysiology: For direct measurement of ion currents through reconstituted c-rings in planar lipid bilayers

The reconstitution process is critical and typically involves:

• Mixing purified protein with appropriate lipids (typically E. coli polar lipids or synthetic mixtures)

• Detergent removal via dialysis, Bio-Beads, or gel filtration

• Verification of proteoliposome formation by dynamic light scattering and electron microscopy

When studying specifically the c-subunit's role, researchers often use a hybrid approach where Shewanella baltica atpE is reconstituted with other ATP synthase subunits from model organisms like E. coli to isolate its specific contribution to enzyme function .

How can researchers investigate the relationship between quorum sensing and ATP synthase activity in Shewanella baltica?

Investigating the quorum sensing-ATP synthase relationship requires integrative approaches:

• Gene Expression Analysis: qRT-PCR and RNA-seq to quantify changes in atpE expression in response to quorum sensing molecules

• Deletion Studies: Creating knockout mutants of luxR-type genes and measuring effects on ATP synthase expression and activity

• Proteomics: Comparative analysis of ATP synthase subunit abundance under different quorum sensing states

• Biofilm Assays: Quantification of biofilm formation while modulating ATP synthase activity

Studies have revealed that Shewanella baltica regulates biofilm formation via quorum sensing, which indirectly involves ATP synthase activity under stress conditions. Deletion of luxR-type genes downregulates biofilm-related genes (pomA), suggesting a link between energy metabolism and biofilm stability.

What advanced biophysical techniques provide insights into the proton translocation mechanism of Shewanella baltica ATP synthase?

Several sophisticated biophysical approaches can elucidate the proton translocation mechanism:

• Cryo-Electron Microscopy: To determine high-resolution structures of the complete ATP synthase complex, revealing the detailed architecture of the c-ring/a-subunit interface

• Hydrogen/Deuterium Exchange Mass Spectrometry: To identify dynamic regions and conformational changes during the catalytic cycle

• Single-Molecule FRET: To measure rotational dynamics and conformational changes in real-time

• Molecular Dynamics Simulations: To model proton movement through the c-ring based on structural data

Similar approaches with E. coli ATP synthase have revealed distinct conformational states during catalysis, particularly involving the ε subunit that engages the α, β, and γ subunits to regulate enzyme function . These techniques could be adapted to investigate species-specific mechanisms in Shewanella baltica.

How does the structure and function of Shewanella baltica atpE compare to other marine bacteria?

Comparative analysis reveals several distinctions in Shewanella baltica atpE:

• Amino Acid Composition: Enriched in acidic residues in surface-exposed regions compared to non-marine bacteria, contributing to halotolerance

• Ion Specificity: While some marine bacteria show Na⁺ coupling, S. baltica maintains H⁺ coupling with modified residues at the ion-binding site

• Oligomeric Structure: The c-ring of S. baltica typically contains 11 subunits, whereas other marine bacteria may have 10-15 subunits

These structural differences reflect adaptations to the psychrotrophic lifestyle of Shewanella baltica in marine environments. Proteomic analyses demonstrate that S. baltica adjusts ATP synthase expression in response to salinity changes, optimizing energy production through modified c-subunit configurations.

What role does the ATP synthase c-subunit play in osmotic stress adaptation in Shewanella baltica?

The ATP synthase c-subunit plays a multifaceted role in osmotic stress adaptation:

• Expression Regulation: Upregulation of atpE gene expression under high salinity conditions

• Structural Modifications: Post-translational modifications that alter c-ring stability under varying ionic strengths

• Proton/Sodium Selectivity: Fine-tuning of ion selectivity to maintain PMF generation in changing salt concentrations

• Energy Conservation: Optimizing ATP production efficiency during osmotic challenge

Proteomic analyses specifically show that S. baltica adjusts ATP synthase expression in response to salinity changes. This adaptation involves DNA-binding proteins and polyamine uptake to stabilize nucleoid structure, with the c-subunit properties being critical to maintain function across osmotic gradients.

How does temperature affect the stability and function of recombinant Shewanella baltica atpE?

As a psychrotrophic organism, S. baltica's ATP synthase displays notable temperature-dependent properties:

• Cold Stability: Recombinant atpE retains significant activity at 4-15°C, unlike mesophilic counterparts

• Thermal Transition Points: Differential scanning calorimetry reveals broader thermal transition ranges (10-40°C) compared to mesophilic proteins

• Catalytic Rate: Lower kcat at reduced temperatures but with higher catalytic efficiency (kcat/Km) compared to mesophilic homologs

• Structural Flexibility: Increased flexibility in loop regions connecting transmembrane helices, facilitating function at lower temperatures

The recombinant subunit retains functional integrity when expressed in E. coli, with activity validated through in vitro assays. Its stability at low temperatures aligns with S. baltica's psychrotrophic nature. This makes the recombinant protein valuable for studying cold adaptation mechanisms in membrane proteins.

How can structural biology approaches be used to study inhibitor binding to Shewanella baltica ATP synthase?

Structural biology provides crucial insights into inhibitor binding:

• Homology Modeling: Building 3D models based on related structures, as demonstrated for other ATP synthase subunits

• Molecular Docking: In silico screening of potential inhibitors against the c-ring structure

• X-ray Crystallography/Cryo-EM: Determining high-resolution structures of inhibitor-bound complexes

• NMR Spectroscopy: Identifying specific residues involved in inhibitor interactions

The methodology for homology modeling would include:

• Template identification and sequence alignment

• Model building using spatial restraints

• Energy minimization and refinement using molecular dynamics simulation

• Validation of model quality using Ramachandran plots, ERRAT, and Verify_3D metrics

Similar approaches with other ATP synthases have identified inhibitors with binding energies between -8.69 and -8.44 kcal/mol , providing starting points for developing Shewanella-specific compounds.

What is the relationship between ATP synthase activity and biofilm formation in Shewanella baltica?

The relationship between ATP synthase and biofilm formation involves several interconnected pathways:

• Energy-Dependent Regulation: ATP availability influences expression of biofilm-associated genes

• Quorum Sensing Integration: ATP synthase activity modulates quorum sensing molecule production

• Membrane Potential Control: The proton-pumping activity affects cell surface properties

• Metabolic Sensing: ATP/ADP ratio serves as a metabolic cue for biofilm developmental stages

Studies specifically reveal that Shewanella baltica regulates biofilm formation via quorum sensing, which indirectly involves ATP synthase activity under stress conditions. Deletion of luxR-type genes downregulates biofilm-related genes (pomA), suggesting a mechanistic link between energy metabolism and biofilm stability.

Research approaches to investigate this relationship include:

• Genetic manipulation of atpE expression levels

• Monitoring biofilm formation under ATP synthase inhibition

• Comparative transcriptomics between planktonic and biofilm states

• Microscopic analysis of biofilm architecture under varying energy states

How can molecular dynamics simulations enhance our understanding of proton translocation through the Shewanella baltica c-ring?

Molecular dynamics (MD) simulations offer powerful insights into proton translocation mechanisms:

• Structural Dynamics: Revealing conformational fluctuations of the c-ring under physiological conditions

• Ion Pathway Mapping: Identifying the complete proton translocation pathway through the a/c interface

• Energetic Barriers: Calculating free energy profiles for proton movement through the complex

• Mutagenesis Prediction: Simulating effects of point mutations on proton translocation efficiency

Methodological approach would include:

• Building a membrane-embedded model of the c-ring and adjacent subunits

• Parameterizing the system with appropriate force fields

• Performing long-timescale simulations (>100 ns) under various proton motive force conditions

• Employing enhanced sampling techniques to capture rare protonation/deprotonation events

Similar approaches with E. coli ATP synthase have revealed distinct conformational states during catalysis , which could guide investigations into Shewanella-specific mechanisms, particularly regarding adaptations to marine environments.

What strategies can resolve expression and purification difficulties with recombinant Shewanella baltica atpE?

Common challenges and their solutions include:

Additionally, researchers should consider:

• Testing different fusion tags beyond His-tag (MBP, SUMO) to enhance solubility

• Optimizing codon usage for E. coli expression

• Exploring cell-free expression systems for difficult constructs

• Using nanodiscs or amphipols as alternatives to detergents for membrane protein stabilization

Post-purification, the protein should be lyophilized and reconstituted in sterile water with glycerol for stability, with quality assurance including SDS-PAGE verification and mass spectrometry analysis to confirm molecular weight and sequence integrity.

How can researchers troubleshoot activity assays when studying recombinant Shewanella baltica ATP synthase components?

Troubleshooting activity assays requires systematic evaluation:

• Buffer Composition Issues:

  • Adjust ionic strength to marine-relevant conditions (0.2-0.5M NaCl)

  • Optimize pH range (7.0-8.0) for maximum activity

  • Test different divalent cations (Mg²⁺, Mn²⁺) as cofactors

• Reconstitution Problems:

  • Vary lipid composition to include marine-relevant phospholipids

  • Optimize protein:lipid ratios (typically 1:50 to 1:200)

  • Test different reconstitution methods (dialysis vs. Bio-Beads)

• Assay-Specific Considerations:

  • For ATP synthesis: Verify coupling enzyme activity independently

  • For proton transport: Calibrate pH indicator response curves

  • For rotational assays: Check fluorophore attachment efficiency

• Environmental Parameters:

  • Test activity across temperature range (4-30°C)

  • Examine salt concentration effects (0-500mM)

  • Consider oxygen levels (aerobic vs. microaerobic)

Similar troubleshooting approaches with E. coli ATP synthase have revealed multiple distinct conformational states during catalysis , which could inform expectations for Shewanella baltica experiments.

What are the key considerations when designing site-directed mutagenesis experiments for Shewanella baltica atpE functional studies?

Effective mutagenesis strategies should consider:

• Target Selection Based on Conservation:

  • Align atpE sequences across diverse species to identify conserved vs. variable residues

  • Focus on species-specific residues that may confer psychrotrophic adaptations

• Functional Domain Targeting:

  • Ion-binding site residues (typically Asp or Glu in transmembrane helix 2)

  • Subunit-subunit interface residues affecting c-ring stability

  • Lipid-interacting residues on the outer surface of the c-ring

• Technical Design Considerations:

  • Use codon optimization for expression host

  • Design primers with appropriate melting temperatures and minimal secondary structure

  • Include silent mutations to introduce diagnostic restriction sites

• Validation Strategy:

  • Compare expression levels between wild-type and mutants

  • Assess structural integrity via CD spectroscopy

  • Measure functional impact through reconstitution and activity assays

Homology modeling approaches similar to those used for other ATP synthase subunits can guide the selection of mutation sites by providing structural context for residue positions and interactions.

How might synthetic biology approaches incorporate Shewanella baltica ATP synthase components for bioenergetic applications?

Emerging synthetic biology applications include:

• Engineered Minimal ATP Synthase Systems:

  • Incorporating S. baltica atpE into simplified ATP synthase constructs

  • Creating hybrid enzymes with components from thermophilic and psychrophilic organisms

  • Developing modular assembly systems for customized ATP synthases

• Nanoscale Power Generation:

  • Integrating c-rings into artificial membrane systems for ATP production

  • Coupling with light-driven proton pumps for solar energy conversion

  • Developing ATP synthase-powered nanomotors using the rotary mechanism

• Biosensing Applications:

  • Utilizing ATP synthase components as sensitive detectors for proton gradients

  • Developing stress-responsive bioenergetic circuits using S. baltica regulatory elements

  • Creating cell-free energy generation systems for biosensor platforms

The application of S. baltica ATP synthase components is particularly promising due to their environmental adaptability, with proven enzymatic stability at low temperatures aligning with the organism's psychrotrophic nature.

What evolutionary insights might be gained from comparative studies of ATP synthase c-subunits across Shewanella species?

Comparative evolutionary analysis could reveal:

• Adaptive Radiation Patterns:

  • Correlation between c-subunit sequence and environmental niches across Shewanella species

  • Identification of convergent evolution events in species from similar habitats

  • Mapping of selection pressures on different functional domains

• Horizontal Gene Transfer Events:

  • Evidence of recombination or gene transfer in ATP synthase operons

  • Integration of foreign genetic elements affecting c-subunit structure or regulation

  • Mosaic evolutionary history of different ATP synthase components

• Structure-Function Relationships:

  • Correlation between amino acid substitutions and functional adaptations

  • Identification of co-evolving residues suggesting functional coupling

  • Reconstruction of ancestral sequences to understand evolutionary trajectories

Such comparative approaches could build upon existing knowledge that S. baltica adjusts ATP synthase expression in response to environmental conditions such as salinity changes, revealing how these adaptive mechanisms evolved across the genus.

How might cryo-EM techniques advance our understanding of the complete Shewanella baltica ATP synthase structure?

Cryo-EM approaches offer promising avenues for structural elucidation:

Similar cryo-EM approaches with E. coli ATP synthase have revealed multiple distinct conformational states during catalysis , providing a methodological framework that could be adapted for Shewanella baltica studies.