Recombinant Shewanella piezotolerans ATP synthase subunit c (atpE)

What is Shewanella piezotolerans atpE and why is it significant for research?

Shewanella piezotolerans atpE encodes the c subunit of ATP synthase, a critical component of the F0 sector that forms the proton-translocating channel within the membrane. This protein is particularly significant because it comes from an organism adapted to deep-sea environments, specifically isolated from sediment in the west Pacific at depths of 1,914 meters . The piezotolerant nature of S. piezotolerans WP3 allows it to grow across pressure ranges from 0.1 to 50 MPa and temperatures from 0 to 35°C, making its membrane proteins particularly interesting for understanding pressure adaptation mechanisms . ATP synthase subunit c is directly involved in coupling proton translocation to ATP synthesis, thereby connecting membrane potential to energy production. For researchers, studying this protein provides insights into bioenergetic adaptations that enable survival in extreme environments, particularly how membrane protein function is maintained under high hydrostatic pressure.

What is the molecular structure and characteristics of the S. piezotolerans atpE protein?

The S. piezotolerans ATP synthase subunit c (atpE) is a small, hydrophobic membrane protein consisting of 83 amino acids with the sequence: METVLGMTAIAVALLIGMGALGTAIGFGLLGGKFLEGAARQPEMAPMLQVKMFIVAGLLDAVTMIGVGIALFMLFTNPLGAML . As a typical c-subunit, it is characterized by its predominantly alpha-helical structure that spans the membrane, with hydrophobic amino acids dominating its composition to facilitate membrane integration. The protein contains glycine-rich motifs that likely provide structural flexibility needed for rotary function within the ATP synthase complex. In recombinant form, the protein is typically produced with an N-terminal His-tag to facilitate purification while maintaining functional integrity . The protein's structure likely reflects adaptations to high-pressure environments, potentially featuring amino acid compositions or structural elements that maintain flexibility and functionality under increased hydrostatic pressure. Based on studies of other Shewanella species, the c-subunit would participate in forming the c-ring of approximately 10-15 subunits in the F0 sector of ATP synthase.

How should recombinant S. piezotolerans atpE protein be stored and handled for optimal stability?

Recombinant S. piezotolerans ATP synthase subunit c requires careful handling to maintain stability due to its hydrophobic nature and membrane protein characteristics. The protein should be stored at -20°C or preferably -80°C for extended storage periods . It is typically provided as a lyophilized powder or in a storage buffer containing Tris/PBS with 6% trehalose (pH 8.0) or in Tris-based buffer with 50% glycerol . When working with the protein, it's critical to avoid repeated freeze-thaw cycles as these can significantly reduce protein stability and activity. For laboratory use, it is recommended to reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and then add glycerol to a final concentration of 5-50% before aliquoting for long-term storage . For working stocks, store aliquots at 4°C for up to one week to minimize degradation. Prior to opening any vial containing the protein, briefly centrifuge to bring contents to the bottom, especially important for lyophilized preparations. For membrane proteins like atpE, consider using mild detergents during reconstitution if functional studies are planned, as this helps maintain the native conformation.

What expression systems are most effective for producing recombinant S. piezotolerans atpE?

E. coli expression systems have proven most effective for producing recombinant S. piezotolerans ATP synthase subunit c, with successful expression documented using N-terminal His-tagged constructs . When designing expression strategies, researchers should consider several critical factors that influence yield and functionality. The highly hydrophobic nature of this membrane protein necessitates careful optimization of induction conditions, including lower temperatures (16-20°C) and reduced inducer concentrations to prevent inclusion body formation. Specialized E. coli strains like C41(DE3) or C43(DE3), developed specifically for membrane protein expression, often provide better results than standard BL21(DE3) strains. For purification, employing a two-step approach combining immobilized metal affinity chromatography (utilizing the His-tag) followed by size exclusion chromatography typically yields the purest protein preparations. The inclusion of appropriate detergents such as n-dodecyl β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) during cell lysis and throughout purification is crucial for maintaining protein solubility and native conformation. When higher yields are required, consider baculovirus-insect cell systems as an alternative expression platform, particularly if post-translational modifications are important for functionality.

How does pressure affect the structure and function of S. piezotolerans ATP synthase subunit c?

The effect of pressure on S. piezotolerans ATP synthase subunit c represents a fascinating aspect of deep-sea adaptation mechanisms. S. piezotolerans WP3 can thrive under pressures ranging from 0.1 to 50 MPa, suggesting that its membrane proteins, including ATP synthase components, maintain functionality across this pressure range . Pressure influences protein structure primarily by affecting protein volume and hydration, with high pressure favoring more compact conformations. For membrane proteins like atpE, pressure effects are particularly complex due to simultaneous compression of the lipid bilayer, which increases in thickness and decreases in fluidity. In S. piezotolerans, the atpE protein likely possesses specific amino acid compositions that confer barotolerance, potentially including higher proportions of small amino acids at key positions to accommodate pressure-induced volume changes without loss of function. The c-ring structure formed by multiple atpE subunits would need to maintain rotational flexibility even under increased pressure to facilitate proton translocation coupled to ATP synthesis. Experimental investigations using high-pressure biophysical techniques, including high-pressure spectroscopy and functional assays in pressure chambers, would be necessary to directly characterize these adaptations.

What methods are most effective for studying the functional properties of recombinant S. piezotolerans atpE?

Investigating the functional properties of recombinant S. piezotolerans ATP synthase subunit c requires specialized techniques that account for its membrane protein nature and potential pressure adaptations. Reconstitution into proteoliposomes represents a primary approach, allowing researchers to assess proton translocation functions under controlled conditions. This can be combined with potentiometric dyes like 9-amino-6-chloro-2-methoxyacridine (ACMA) to measure proton gradient formation. For deeper functional characterization, collaborative assembly with other ATP synthase subunits to form partial or complete complexes allows assessment of the c-ring's rotational properties and coupling to ATP synthesis. Membrane potential measurements using fluorescent dyes such as thioflavin T (ThT) can reveal connections between ATP synthase activity and membrane energization, as demonstrated in studies with Shewanella oneidensis showing the link between membrane potential and extracellular electron transfer . For pressure-related studies, specialized high-pressure chambers compatible with spectroscopic measurements enable real-time monitoring of structural changes or activity under variable pressure conditions. Advanced techniques like hydrogen-deuterium exchange mass spectrometry under pressure can identify specific regions of the protein that undergo conformational changes in response to pressure variation.

How does the function of ATP synthase subunit c in S. piezotolerans compare with other Shewanella species from different depth environments?

Comparative analysis of ATP synthase subunit c across Shewanella species from varying depths reveals evolutionary adaptations to different pressure regimes. Unlike shallow-water Shewanella species, S. piezotolerans WP3 demonstrates growth capabilities under pressures up to 50 MPa, suggesting significant adaptations in its membrane proteins, including ATP synthase components . These adaptations likely manifest as amino acid substitutions at key positions that maintain protein flexibility and function under compression. Deep-sea adaptations in membrane proteins often involve increased proportions of small amino acids like glycine and alanine, reduced hydrophobicity in transmembrane segments, and modifications that optimize protein-lipid interactions under pressure. The c-ring stoichiometry (number of c subunits per ring) may also differ between shallow and deep-water species, potentially affecting the bioenergetic efficiency of ATP synthesis. Similar to observed pressure adaptations in nitrate reductase systems, where S. piezotolerans maintains two isoforms (NAP-α and NAP-β) with different pressure responses, ATP synthase components may show specialized pressure tolerance mechanisms . Specifically, the NAP-α system demonstrated higher tolerance to elevated pressure in S. piezotolerans, suggesting analogous adaptations might exist in ATP synthase components to maintain energy production under deep-sea conditions.

What is the relationship between ATP synthase activity and extracellular electron transfer in Shewanella species?

The relationship between ATP synthase activity and extracellular electron transfer (EET) in Shewanella species represents a critical intersection of bioenergetic pathways. In Shewanella oneidensis, membrane potential has been directly linked to EET capabilities, with studies demonstrating that the inner membrane becomes hyperpolarized during electron transfer processes . This hyperpolarization results from proton translocation during the redox cycling of the quinone pool, where quinones are reduced by formate dehydrogenase and lactate dehydrogenase, and subsequently oxidized by CymA . The membrane potential generated contributes to the proton motive force that drives ATP synthesis via the F1F0-ATP synthase, of which the c subunit (atpE) forms an essential component. During EET, electrons are transported from CymA through the Mtr pathway to extracellular acceptors, creating a link between the outer membrane electron transfer and inner membrane energization. This connection suggests that the ATP synthase c subunit plays an indirect but crucial role in supporting EET capabilities by participating in energy conservation from the generated proton gradient. Furthermore, recent studies show that while utilizing high-potential electron acceptors, Shewanella employs proton- and sodium-pumping NADH dehydrogenases that further hyperpolarize the membrane, creating additional driving force for ATP synthesis .

How can recombinant S. piezotolerans atpE be used to study pressure adaptation mechanisms?

Recombinant S. piezotolerans ATP synthase subunit c provides an excellent model system for investigating molecular mechanisms of pressure adaptation in deep-sea organisms. Researchers can employ comparative structural studies between atpE from S. piezotolerans and homologs from shallow-water Shewanella species to identify specific amino acid substitutions that confer barotolerance. By creating chimeric proteins or site-directed mutants where key residues are exchanged between pressure-sensitive and pressure-tolerant homologs, the specific contributions of individual amino acids to pressure adaptation can be assessed. Functional assays under varying pressure conditions, using high-pressure chambers coupled to activity measurements, can reveal how the protein maintains proper folding and function despite increased hydrostatic pressure. Molecular dynamics simulations incorporating pressure parameters offer complementary computational approaches to understand conformational dynamics under pressure. Reconstitution experiments comparing protein function in liposomes composed of different lipid compositions can further illuminate how protein-lipid interactions contribute to pressure tolerance, particularly important since S. piezotolerans WP3 has been shown to modulate its membrane composition in response to pressure changes . These approaches collectively can reveal fundamental principles of protein adaptation to extreme environments.

What methodological challenges exist when studying recombinant S. piezotolerans atpE and how can they be overcome?

Research with recombinant S. piezotolerans ATP synthase subunit c presents several significant methodological challenges that require specialized approaches. The primary challenge stems from its highly hydrophobic nature as a membrane protein, leading to poor solubility and potential misfolding during recombinant expression. To overcome this, researchers should optimize expression conditions using specialized E. coli strains designed for membrane proteins and employ fusion partners like MBP (maltose-binding protein) to enhance solubility. Purification protocols require careful detergent selection, with mild non-ionic detergents like DDM (n-dodecyl β-D-maltoside) often providing the best balance between protein extraction and native structure preservation. For functional studies, reconstitution into liposomes presents another challenge, particularly achieving consistent orientation of the protein within the membrane. Controlled reconstitution protocols using detergent removal by dialysis or biobeads can improve consistency. Studies under high pressure require specialized equipment capable of maintaining pressure while allowing spectroscopic or functional measurements, which can be addressed using custom-designed high-pressure cells compatible with spectrophotometers or activity assays. For structural studies, traditional crystallography is challenging with membrane proteins, making newer approaches like cryo-electron microscopy particularly valuable, especially when coupled with lipid nanodiscs to maintain a native-like membrane environment.

What insights can comparative genomics and proteomics provide about S. piezotolerans atpE evolution and function?

Comparative genomics and proteomics approaches offer powerful insights into the evolutionary trajectory and functional specialization of S. piezotolerans ATP synthase subunit c. Sequence analysis across Shewanella species from various depths reveals signatures of selective pressure associated with depth adaptation, potentially identifying positions under positive selection that contribute to barotolerance. Whole-genome analyses can place atpE evolution in the broader context of adaptation to the deep-sea environment, revealing whether horizontal gene transfer contributed to acquisition of pressure-tolerant versions or if adaptations emerged gradually through vertical inheritance. Synteny analysis of the ATP synthase operon structure across species can identify conservation patterns or rearrangements that might relate to regulatory adaptations for different pressure regimes. Proteomic approaches comparing protein expression profiles under different pressure conditions can reveal whether S. piezotolerans modulates ATP synthase abundance in response to pressure changes, similar to observed differential expression patterns in its nitrate reduction systems where pressure-induced expression has been documented . Post-translational modification analyses may uncover pressure-specific modifications that contribute to protein stability or function. Interactome studies can identify pressure-specific protein-protein interactions that might stabilize the ATP synthase complex under high hydrostatic pressure conditions.

How can researchers design experiments to investigate the role of S. piezotolerans atpE in pressure-regulated bioenergetics?

Designing experiments to investigate S. piezotolerans atpE's role in pressure-regulated bioenergetics requires a multifaceted approach combining genetic, biochemical, and biophysical methods. Researchers should first establish pressure-adjustable cultivation systems similar to those described for S. piezotolerans WP3 growth studies, where cultures can be maintained in pressure vessels at precisely controlled hydrostatic pressures ranging from 0.1 to 50 MPa . Gene expression analysis using qRT-PCR or RNA-seq across pressure gradients can quantify atpE transcriptional responses to pressure changes, while western blotting with specific antibodies can confirm corresponding protein level changes. For functional studies, membrane vesicles isolated from cells grown at different pressures can be used to measure ATP synthesis rates and proton pumping activity. Direct measurement of membrane potential using fluorescent dyes like thioflavin T (ThT) under pressure conditions, as demonstrated for monitoring Shewanella membrane potential responses , provides insights into bioenergetic states. Construction of atpE knockout strains complemented with various versions of the gene (wild-type, pressure-sensitive homologs from shallow-water species, or site-directed mutants) allows assessment of specific residues' contributions to pressure tolerance. Proteoliposome reconstitution experiments conducted under variable pressure can directly measure how pressure affects the proton translocation function of the c-subunit in isolation from other cellular components.

What statistical approaches are most appropriate for analyzing pressure-dependent changes in atpE structure and function?

Statistical analysis of pressure-dependent changes in S. piezotolerans ATP synthase subunit c structure and function requires specialized approaches that account for the unique characteristics of pressure response data. For continuous measurements across pressure gradients, nonlinear regression models often better capture the relationship between pressure and functional parameters than linear models, as biological responses to pressure frequently show threshold effects or saturation. When comparing discrete pressure points, repeated measures ANOVA with appropriate post-hoc tests should be employed to account for within-sample correlations. Statistical power calculations should consider that pressure effects may be subtle, requiring larger sample sizes to detect significant differences. For structural studies generating large datasets (such as HDX-MS or molecular dynamics simulations), multivariate statistical approaches including principal component analysis can help identify regions of the protein most affected by pressure changes. Time-series data from pressure adaptation experiments should be analyzed using mixed-effects models that can separate acute pressure responses from adaptive changes. When comparing homologs from different species or mutant variants, two-way ANOVA designs can simultaneously assess the effects of pressure and sequence variation, with particular attention to interaction terms that may indicate differential pressure sensitivity. Bayesian statistical approaches can be particularly valuable when incorporating prior knowledge about protein physics under pressure into the analysis framework.

What interdisciplinary approaches would advance our understanding of S. piezotolerans atpE function in deep-sea environments?

Advancing our understanding of S. piezotolerans ATP synthase subunit c function in deep-sea environments requires innovative interdisciplinary approaches combining expertise from multiple scientific domains. Collaborative efforts between structural biologists and high-pressure physicists could develop improved methods for determining membrane protein structures under pressure, potentially using diamond anvil cells coupled with advanced spectroscopic techniques. Biophysicists and engineers might develop miniaturized pressure chambers compatible with live-cell imaging to visualize ATP synthase dynamics in real-time under different pressure regimes. Environmental microbiologists and oceanographers could design sampling strategies to collect and preserve deep-sea Shewanella species from various depths, allowing more comprehensive comparative analyses across pressure gradients. Computational biologists and bioinformaticians could apply machine learning approaches to identify subtle sequence patterns associated with pressure adaptation across large datasets of c-subunit sequences from diverse marine environments. Synthetic biologists might engineer chimeric ATP synthase complexes incorporating modules from pressure-adapted and pressure-sensitive species to test specific hypotheses about functional domains. Systems biologists could integrate multi-omics data to place ATP synthase function within the broader context of cellular pressure responses, similar to approaches used to study S. piezotolerans nitrate reduction systems under high hydrostatic pressure . Biochemical engineers might develop biomimetic systems based on pressure-adapted ATP synthases for energy generation applications in high-pressure industrial processes.

What emerging technologies might enhance research on S. piezotolerans atpE and its role in pressure adaptation?

Emerging technologies offer exciting possibilities for advancing research on S. piezotolerans ATP synthase subunit c and its role in pressure adaptation. Cryo-electron microscopy (cryo-EM) with improved resolution capabilities could reveal structural details of the ATP synthase complex under pressure-mimicking conditions when combined with specialized sample preparation techniques. Single-molecule force spectroscopy using atomic force microscopy could directly measure how pressure affects the mechanical stability and conformational states of individual atpE proteins or assembled c-rings. Microfluidic high-pressure chambers with integrated sensing capabilities would allow rapid screening of protein variants under varying pressure conditions to identify key residues contributing to barotolerance. Advanced computational approaches, including quantum mechanics/molecular mechanics (QM/MM) simulations, could model proton translocation through the c-ring under different pressure regimes with unprecedented detail. CRISPR-Cas9 genome editing techniques adapted for Shewanella could enable precise modification of atpE in its native context to test functional hypotheses about pressure adaptation. Mass spectrometry innovations, particularly hydrogen-deuterium exchange mass spectrometry (HDX-MS) compatible with high-pressure conditions, could map pressure-induced conformational changes with amino acid resolution. Nanopore-based single-molecule sensing might enable real-time monitoring of individual c-subunit incorporation into membranes under pressure. Time-resolved serial crystallography at X-ray free-electron lasers represents another frontier technology that could capture dynamic structural changes in the protein during function under variable pressure conditions.