Recombinant Shewanella pealeana ATP synthase subunit c (atpE)

What is Shewanella pealeana ATP synthase subunit c and what is its significance in bioenergetics research?

Shewanella pealeana ATP synthase subunit c (atpE) is a critical component of the F0 sector of ATP synthase, functioning as part of the membrane-embedded proton channel that drives ATP synthesis. This protein, encoded by the atpE gene (locus Spea_4245), consists of 85 amino acids and forms part of the c-ring structure essential for energy transduction in this organism . The significance of this protein lies in understanding bioenergetic processes in Shewanella species, which are notable for their diverse respiratory capabilities, particularly their ability to utilize a broad range of electron acceptors under anaerobic conditions . Shewanella pealeana was originally isolated from the accessory nidamental gland of the squid Loligo pealei and has been characterized as a mesophilic, facultatively anaerobic, psychrotolerant bacterium with optimal growth at 25-30°C and pH 6.5-7.5 .

How does the structure of ATP synthase subunit c from Shewanella pealeana compare to that of other organisms?

The ATP synthase subunit c from Shewanella pealeana features a highly conserved structural motif consisting primarily of alpha-helical secondary structure, similar to those observed in other organisms . The protein has a sequence of 85 amino acids (METVISFTAIAVAIMIGLAALGTGIGFAILGGKFLEASARQPELAPALQTKMFIVAGLLDAISMIAVGVALFFVFANPFLGQLAG) , which forms membrane-spanning helices that assemble into the c-ring of ATP synthase. While the fundamental structure is conserved across species, notable differences exist in c-ring stoichiometry among organisms, with known variations ranging from c10 to c15 subunits per ring . This stoichiometric variation directly affects the ion-to-ATP coupling ratio, which ranges from 3.3 to 5.0 among studied organisms, presenting a significant area for comparative research .

How does the c-subunit contribute to energy conservation strategies in Shewanella pealeana under anaerobic conditions?

The c-subunit of ATP synthase in Shewanella pealeana forms an integral part of a sophisticated energy conservation strategy under anaerobic conditions. Genomic and metabolic modeling research on Shewanella species has revealed that, contrary to expectations for respiratory organisms, Shewanella primarily uses substrate-level phosphorylation via enzymes like acetate kinase (AckA) for ATP production under anaerobic conditions, while ATP synthase plays a secondary role . Under these conditions, the c-ring of ATP synthase may function bidirectionally, either contributing minimally to ATP synthesis or operating in reverse as a proton pump that generates proton motive force (PMF) .

This adaptability is particularly significant because it allows Shewanella to maintain energy homeostasis when utilizing diverse terminal electron acceptors including metals, nitrate, and sulfur compounds. The flexible operation of the c-ring enables the cell to balance redox reactions, PMF generation, and ATP production through a complex interplay of electron transport chain components, including formate dehydrogenase and NADH dehydrogenase, whose activities are coupled with terminal electron acceptor reduction . This sophisticated management of energy resources may contribute to Shewanella pealeana's ecological success in marine sediments where electron acceptor availability fluctuates.

What is the relationship between c-subunit structure and proton leak phenomena in ATP synthase?

Recent research has identified a critical relationship between ATP synthase c-subunit structure and proton leak phenomena that significantly impacts cellular bioenergetics. While not specifically studied in Shewanella pealeana, investigations of ATP synthase c-subunits across species have revealed that the c-subunit ring can mediate a regulated proton leak that influences inner membrane efficiency and ATP production . This leak function appears to be an intrinsic property of the c-ring structure, potentially involving conformational changes that create transient proton-conducting pathways .

The leak through the c-subunit assembly has been implicated in the formation or regulation of the mitochondrial permeability transition pore (mPTP), a channel spanning the inner membrane that regulates cellular processes including development and cell death . Abnormal elevation of c-subunit levels has been associated with persistence of immature metabolic phenotypes characterized by increased membrane leak, termed "leak metabolism" . This relationship suggests that precise regulation of c-subunit expression and assembly is crucial for maintaining proper cellular bioenergetics and development. The study of c-subunit leak phenomena in Shewanella pealeana could provide valuable insights into how this organism balances energy efficiency with metabolic flexibility in its natural environment.

What are the optimal protocols for recombinant expression and purification of Shewanella pealeana ATP synthase subunit c?

Based on successful approaches with other ATP synthase c-subunits, the following optimized protocol is recommended for Shewanella pealeana atpE:

Expression System Selection:
The recombinant expression of Shewanella pealeana ATP synthase subunit c is optimally achieved using an Escherichia coli expression system with codon optimization . Three vector systems have demonstrated utility for c-subunit expression: pMAL-c2x, pET-32a(+), and pFLAG-MAC, with the pMAL-c2x system (producing a maltose-binding protein fusion) showing superior results for stability and yield .

Cloning and Expression Process:

• Design a synthetic atpE gene with E. coli optimized codons based on the known amino acid sequence

• Include appropriate restriction sites for directional cloning into the selected vector

• Transform the expression construct into an appropriate E. coli strain (BL21(DE3) or similar)

• Induce protein expression using IPTG (typically 1.0 mM) at mid-log phase

• Allow expression to proceed for 30 minutes to minimize potential toxicity issues

Purification Strategy:
The high hydrophobicity of the c-subunit necessitates specialized purification approaches:

• Harvest cells and prepare lysate in the presence of protease inhibitors

• Initial purification using affinity chromatography based on the fusion tag

• For MBP-fusion proteins, use amylose resin with elution by maltose

• Consider tag removal with appropriate protease if needed for downstream applications

• Further purification may include size exclusion chromatography in the presence of appropriate detergents to maintain protein solubility

• Verify purification by SDS-PAGE and western blotting using anti-c-subunit antibodies

Successful expression and purification should yield protein suitable for structural studies, reconstitution experiments, and functional analyses.

What methods are recommended for studying c-ring assembly and stoichiometry in Shewanella pealeana?

Investigating c-ring assembly and stoichiometry in Shewanella pealeana requires a multi-faceted approach combining biochemical, structural, and computational techniques:

1. Isolation and Purification of Intact c-rings:

• Extract membrane fractions from Shewanella pealeana cells using differential centrifugation

• Solubilize membranes with appropriate detergents (DDM, C12E8, or similar)

• Perform blue native PAGE to preserve native complex structure

• Confirm complex integrity using western blotting with anti-c-subunit antibodies

2. Structural Analysis Methods:

• Atomic Force Microscopy (AFM) to directly visualize c-ring structures

• Cryo-electron microscopy for high-resolution structural determination

• X-ray crystallography if crystals can be obtained

• Cross-linking mass spectrometry to determine subunit arrangements and interactions

3. Stoichiometry Determination Approaches:

• Mass spectrometry of intact c-rings

• Quantitative amino acid analysis

• Cross-linking and SDS-PAGE gel shift assays

• Fluorescence resonance energy transfer (FRET) between labeled c-subunits

4. Reconstitution Experiments:

• In vitro assembly of c-rings from purified recombinant c-subunits

• Incorporation of c-rings into liposomes for functional studies

• Assessment of proton translocation using pH-sensitive dyes or electrodes

5. Computational Modeling:

• Molecular dynamics simulations to predict stable c-ring configurations

• Comparative genomics to identify conserved features impacting stoichiometry

• Genome-scale metabolic modeling to predict energetic consequences of different c-ring stoichiometries

The combination of these approaches can provide comprehensive insights into the assembly, structure, and functional implications of c-ring stoichiometry in Shewanella pealeana.

How can researchers investigate the relationship between ATP synthase activity and electron transport chain function in Shewanella pealeana?

Investigating the relationship between ATP synthase and electron transport chain (ETC) function in Shewanella pealeana requires integrative approaches that span molecular, cellular, and systems biology:

Respiratory Phenotyping:

• Growth characterization using different electron acceptors (O₂, nitrate, fumarate, Fe(III), sulfur compounds) with measurement of substrate depletion rates and biomass yields

• Oxygen consumption rates using high-resolution respirometry

• Membrane potential measurements using fluorescent probes like DiOC₂(3) or tetramethylrhodamine methyl ester

ATP Synthase Activity Assays:

• Measurement of ATP synthesis rates in inverted membrane vesicles

• Proton pumping assays using pH-sensitive fluorescent probes

• ATPase activity measurement using colorimetric phosphate release assays

• In situ ATP synthase activity using luciferase-based bioluminescence assays

Integrative Approaches:

• Generation of atpE conditional expression strains to control ATP synthase levels

• Construction of F₁F₀ ATP synthase point mutants with altered coupling efficiency

• Real-time metabolite profiling using NMR or mass spectrometry

• Isotope labeling experiments to track carbon and electron flow

Systems Biology Approaches:

• Genome-scale metabolic modeling to predict ATP synthase flux under different conditions

• Flux balance analysis to assess the relative contributions of oxidative phosphorylation and substrate-level phosphorylation

• Transcriptomic and proteomic profiling to identify regulatory relationships between ATP synthase and ETC components

This comprehensive approach can reveal how Shewanella pealeana coordinates ATP synthase activity with electron transport chain function across different growth conditions, particularly highlighting the shift between oxidative phosphorylation and substrate-level phosphorylation under aerobic versus anaerobic conditions.

What is the relationship between c-subunit structure and membrane leak phenomena in ATP synthase across different species?

Recent research has established a complex relationship between ATP synthase c-subunit structure and membrane leak phenomena that appears to be conserved across diverse species:

Leak Function Evolution:
While not specifically studied in Shewanella pealeana, investigation of ATP synthase c-subunits across species has revealed that the c-subunit ring can mediate a regulated proton leak that influences membrane efficiency and energy conservation . This leak function appears to be an ancient and conserved property of the c-ring structure, suggesting it may serve important physiological roles beyond ATP synthesis.

Structural Determinants of Leak:
Key structural features that may influence leak properties include:

• The tight packing of c-subunits within the ring

• Specific residues at the interface between adjacent c-subunits

• Interactions between the c-ring and other membrane components

• Lipid composition of the surrounding membrane

Physiological Implications:
The c-subunit leak has been implicated in several important cellular processes:

• Regulation of membrane potential to prevent over-energization

• Thermogenesis in certain specialized tissues

• Contribution to the mitochondrial permeability transition pore

• Modulation of cellular development and differentiation

Abnormal regulation of this leak function has been associated with pathological conditions in higher organisms, including neurodevelopmental disorders . In bacterial systems like Shewanella pealeana, the leak may serve as an important regulatory mechanism for balancing energy production with other cellular needs, particularly during transitions between different electron acceptors or environmental conditions.

How can recombinant Shewanella pealeana ATP synthase c-subunit be used in the study of c-ring assembly and stoichiometry determination?

Recombinant Shewanella pealeana ATP synthase c-subunit provides a valuable tool for investigating fundamental questions about c-ring assembly and stoichiometry through several innovative approaches:

In Vitro Reconstitution Studies:
Purified recombinant c-subunits can be used to:

• Establish controlled assembly conditions to determine factors influencing ring formation

• Investigate the kinetics and thermodynamics of c-ring assembly

• Explore the effects of lipid composition on ring stability and stoichiometry

• Test the impact of mutations on assembly efficiency and final structure

Hybrid Ring Construction:
Recombinant technology enables the creation of:

• Fluorescently labeled c-subunits for visualization of assembly processes

• Chimeric c-subunits combining features from different species

• Co-expression systems to produce rings with defined subunit ratios

• Site-specific crosslinkable c-subunits to capture assembly intermediates

Biophysical Analysis:
With sufficient quantities of purified protein, researchers can perform:

• Advanced structural studies using X-ray crystallography or cryo-EM

• Mass spectrometry of intact rings to determine precise stoichiometry

• Atomic force microscopy to visualize ring topography and dimensions

• Spectroscopic techniques to monitor structural changes during assembly

Functional Reconstitution:
The recombinant protein enables:

• Incorporation of c-rings into liposomes for proton transport studies

• Measurement of passive proton leak through the assembled ring

• Assembly of complete ATP synthase complexes with defined c-ring composition

• Testing of hypotheses about the relationship between stoichiometry and function

This research area is particularly significant as it addresses fundamental questions about the evolution of bioenergetic efficiency across different species and environments, potentially revealing how Shewanella pealeana has optimized its energy conservation strategy for its ecological niche.

What are the major challenges in expressing and purifying functional recombinant ATP synthase c-subunit from Shewanella pealeana?

Expression and purification of functional recombinant ATP synthase c-subunit from Shewanella pealeana presents several significant challenges that researchers must address:

Expression Challenges:

• Extreme hydrophobicity - The c-subunit contains multiple membrane-spanning helices, making it difficult to express in soluble form

• Potential toxicity - Overexpression may disrupt host cell membrane integrity, limiting yield

• Codon usage bias - Differences between Shewanella and expression host codon preferences may reduce expression efficiency

• Proper folding - The α-helical structure requires appropriate chaperone systems for correct folding

Purification Challenges:

• Detergent selection - Identifying detergents that maintain native structure while efficiently solubilizing the protein

• Aggregation tendencies - Preventing non-specific aggregation during concentration steps

• Tag interference - Fusion tags may affect structure or oligomerization properties

• Maintaining stability - Preventing degradation during purification process

Functional Assessment Challenges:

• Native conformation verification - Confirming that recombinant protein adopts the correct α-helical structure

• Oligomerization capability - Determining if purified protein can assemble into c-rings

• Proton conductance - Testing if assembled rings maintain proton transport capability

• Integration with other ATP synthase components - Assessing interaction with a and b subunits

Addressing these challenges requires methodological innovations such as specialized fusion partners (like maltose-binding protein), careful optimization of expression conditions, and the development of appropriate functional assays to verify biological activity .

How might the study of Shewanella pealeana ATP synthase c-subunit contribute to understanding bioenergetic adaptations in extreme environments?

The study of Shewanella pealeana ATP synthase c-subunit offers valuable insights into bioenergetic adaptations to extreme environments, particularly marine and low-temperature settings:

Psychrotolerance Adaptations:
Shewanella pealeana is a psychrotolerant organism isolated from a squid accessory nidamental gland . Its ATP synthase c-subunit likely contains adaptations that maintain flexibility and function at low temperatures, which could reveal molecular mechanisms for cold adaptation in membrane proteins, including:

• Amino acid substitutions that increase structural flexibility

• Modifications that maintain proton conductance at lower temperatures

• Adaptations that preserve c-ring assembly in cold environments

Marine Environment Adaptations:
As a marine bacterium adapted to moderate salinity (optimal growth at 0.5 M NaCl) , S. pealeana ATP synthase may feature:

• Surface residue adaptations for salt tolerance

• Modifications to proton-binding sites to function in marine pH conditions

• Structural features that maintain function under osmotic stress

Metabolic Versatility Insights:
Shewanella pealeana can grow using diverse electron acceptors including oxygen, nitrate, fumarate, iron, manganese, and elemental sulfur . This versatility suggests the ATP synthase may have evolved specific features to:

• Function efficiently during transitions between electron acceptors

• Potentially operate in reverse direction to generate PMF under certain conditions

• Interact dynamically with diverse electron transport chain components

Understanding these adaptations could inform:

• Biotechnological applications requiring enzyme function in extreme conditions

• Evolutionary models of membrane protein adaptation

• Bioenergetic strategies in diverse marine microorganisms

• Biomimetic approaches to creating energy-efficient synthetic systems

What future research directions could advance our understanding of the role of ATP synthase c-subunit in Shewanella pealeana's energy metabolism?

Several promising research directions could significantly advance our understanding of ATP synthase c-subunit function in Shewanella pealeana's energy metabolism:

Structural Biology Approaches:

• High-resolution structural determination of the complete Shewanella pealeana ATP synthase complex

• Comparative structural analysis of the c-ring under different environmental conditions (temperature, pH, salinity)

• Investigation of c-subunit interactions with other ATP synthase components, particularly the a-subunit

• Nanoscale dynamics studies to capture conformational changes during operation

Systems Biology Integration:

• Expansion of genome-scale metabolic models to incorporate detailed ATP synthase function

• Multi-omics approaches to identify regulatory networks controlling ATP synthase expression

• In silico prediction of energetic consequences of different c-ring stoichiometries

• Ecological modeling of energy conservation strategies in natural habitats

Innovative Functional Studies:

• Development of c-subunit variants with altered proton specificity or conductance

• Investigation of c-ring leak phenomena and its physiological significance

• Single-molecule studies of c-ring rotation in reconstituted systems

• Analysis of ATP synthase directionality switching mechanisms under different conditions

Applied Research Potential:

• Engineering optimized ATP synthases for biotechnological applications

• Development of inhibitors targeting specific aspects of ATP synthase function

• Bioelectrochemical systems utilizing Shewanella's electron transport capabilities

• Biomimetic energy conversion systems inspired by Shewanella's adaptations

Progress in these areas would not only illuminate the specific adaptations of Shewanella pealeana's ATP synthase but could also provide broader insights into the evolution of bioenergetic systems and their role in microbial adaptation to diverse environmental niches.

Table 1: Comparative Analysis of ATP Synthase c-Subunit Properties Across Selected Organisms

This table highlights the diversity in c-subunit properties across different organisms, suggesting adaptation to specific ecological niches and energetic requirements. The variation in c-ring stoichiometry directly affects the ion-to-ATP ratio and therefore the bioenergetic efficiency of the organism . Shewanella pealeana's unique adaptations to marine environments and its metabolic versatility make its ATP synthase c-subunit a valuable subject for comparative bioenergetic studies.

Table 2: Experimental Approaches for ATP Synthase c-Subunit Research in Shewanella pealeana

This table provides a roadmap for researchers investigating different aspects of ATP synthase c-subunit function in Shewanella pealeana, outlining methodological approaches, technical requirements, expected outcomes, and anticipated challenges for each research question.