Recombinant Shewanella baltica ATP synthase subunit alpha (atpA), partial

What is Shewanella baltica ATP synthase subunit alpha and what is its function?

Shewanella baltica ATP synthase subunit alpha (atpA) is a critical component of the F1 sector of the bacterial ATP synthase complex. The protein plays an essential role in the catalytic mechanism of ATP synthesis, participating in the conversion of electrochemical ion gradients into chemical energy in the form of ATP. The atpA subunit forms part of the hexameric structure with alternating α and β subunits that constitute the catalytic core of the F1 portion of ATP synthase .

In Shewanella baltica specifically, this protein functions within the context of this facultative anaerobe's diverse respiratory capabilities. The ATP synthase subunit alpha has the EC number 3.6.3.14 and is alternatively known as ATP synthase F1 sector subunit alpha or F-ATPase subunit alpha, reflecting its structural and functional role in the enzyme complex . The recombinant partial version provides researchers with access to a specific portion of the native protein structure for focused studies.

What are the optimal storage and handling conditions for Recombinant Shewanella baltica ATP synthase subunit alpha?

For optimal stability and activity, Recombinant Shewanella baltica ATP synthase subunit alpha should be stored at -20°C, and for extended storage periods, conservation at -20°C or -80°C is recommended . This temperature range helps prevent protein degradation and maintains structural integrity essential for experimental applications.

When working with the recombinant protein, it's important to note that repeated freezing and thawing cycles can significantly compromise protein quality. To avoid this issue, researchers should prepare working aliquots that can be stored at 4°C for up to one week . This approach minimizes freeze-thaw cycles while ensuring convenient access to fresh protein for ongoing experiments.

Before opening the protein vial, brief centrifugation is recommended to bring the contents to the bottom of the container, preventing potential loss of material . This simple precaution is particularly important for lyophilized preparations or solutions with small volumes.

What reconstitution protocols are recommended for the recombinant protein?

The recommended reconstitution protocol for Recombinant Shewanella baltica ATP synthase subunit alpha involves dissolving the protein in deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL . This concentration range provides suitable working solutions for most experimental applications while maintaining protein stability.

For long-term storage of reconstituted protein, the addition of 5-50% glycerol (final concentration) is strongly recommended . Glycerol acts as a cryoprotectant, preventing damage to the protein structure during freezing by inhibiting ice crystal formation. The manufacturer's default final concentration of 50% glycerol can serve as a reference point, but researchers may adjust this based on specific experimental requirements .

After reconstitution with the appropriate glycerol concentration, the solution should be aliquoted to minimize freeze-thaw cycles and stored at -20°C or -80°C for optimal preservation of protein activity and structure.

What is the expected shelf life of the recombinant protein under different storage conditions?

The shelf life of Recombinant Shewanella baltica ATP synthase subunit alpha varies depending on its physical state and storage conditions. In liquid form, the protein typically maintains its stability for approximately 6 months when stored at -20°C or -80°C . This timeframe assumes proper storage conditions with minimal temperature fluctuations.

In lyophilized form, the protein demonstrates enhanced stability with a shelf life of approximately 12 months at -20°C or -80°C . The removal of water in the lyophilization process significantly reduces the potential for degradative reactions, extending the viable storage period for research applications.

It's important to note that the actual shelf life can be influenced by various factors including buffer ingredients, storage temperature consistency, and the inherent stability of the protein itself . For critical experiments, researchers should consider validating protein activity after extended storage periods to ensure experimental reliability.

How can the purity and activity of the recombinant protein be verified in laboratory settings?

Verifying the purity and activity of Recombinant Shewanella baltica ATP synthase subunit alpha is essential for ensuring reliable experimental results. According to product specifications, the recombinant protein should have a purity level exceeding 85% as determined by SDS-PAGE analysis . Researchers can perform their own SDS-PAGE verification to confirm this purity level before proceeding with experiments.

For activity assessment, functional assays relevant to ATP synthase can be employed. ATP hydrolysis assays measuring the release of inorganic phosphate from ATP or coupled enzyme assays monitoring ATP synthesis are standard approaches for ATP synthase activity verification. These assays can be adapted from protocols used for other bacterial ATP synthases, with appropriate modifications for the specific properties of the Shewanella baltica protein .

Additionally, researchers can consider immunological methods for verification. Western blotting techniques using antibodies specific to ATP synthase subunit alpha, similar to those described for plant AtpA , may be adapted for the Shewanella baltica protein with appropriate modifications to account for species-specific differences.

What experimental conditions are optimal for studying ATP synthesis using reconstituted systems with this recombinant protein?

Optimal experimental conditions for studying ATP synthesis using Recombinant Shewanella baltica ATP synthase subunit alpha should be designed based on the physiological context of this facultative anaerobe and the biochemical requirements of ATP synthase function. While specific conditions for Shewanella baltica ATP synthase are not directly provided in the available literature, insights can be drawn from studies on related ATP synthases.

For reconstitution experiments, researchers should consider embedding the complete ATP synthase complex containing the recombinant alpha subunit into proteoliposomes with defined lipid composition. Based on protocols used for other bacterial ATP synthases, these proteoliposomes should be prepared with controlled internal and external ion concentrations to establish the necessary electrochemical gradients for driving ATP synthesis .

The optimal pH range likely falls between 7.0-8.0, reflecting the physiological conditions for Shewanella species. Temperature conditions would typically be set between 25-30°C, corresponding to the mesophilic nature of Shewanella baltica. For generating the ion gradient necessary for ATP synthesis, researchers might employ potassium diffusion potentials using valinomycin, as demonstrated with other ATP synthases . This approach can create an electrical field (Δψ) of approximately 160 mV, which can drive ATP synthesis when combined with an appropriate ion concentration gradient.

How can researchers determine the ion specificity of Shewanella baltica ATP synthase?

Determining the ion specificity of Shewanella baltica ATP synthase is crucial for understanding its bioenergetic mechanism and for designing appropriate experimental protocols. To investigate whether the enzyme preferentially utilizes H+ or Na+ as coupling ions, researchers should implement a systematic experimental approach.

The experimental design should include reconstituted proteoliposomes containing the ATP synthase complex with the recombinant alpha subunit. By selectively establishing H+ or Na+ gradients across the liposomal membrane and measuring ATP synthesis rates, researchers can determine which ion drives the enzyme more efficiently. This can be achieved by preparing proteoliposomes with different internal and external ion compositions and pH values .

Quantitative assessment should include determining threshold values of different driving forces (ΔμNa+, ΔμH+, Δψ) required for ATP synthesis, similar to analyses performed with ATP synthases from other bacteria . These threshold values provide important insights into the energetic requirements of the enzyme and its physiological operation in the bacterial cell.

What methodological approaches are most effective for studying structure-function relationships in ATP synthase subunit alpha?

Investigating structure-function relationships of Shewanella baltica ATP synthase subunit alpha requires an integrated approach combining structural determination, site-directed mutagenesis, and functional assays. This multifaceted strategy enables researchers to correlate specific structural features with their functional consequences.

For structural determination, X-ray crystallography and cryo-electron microscopy represent powerful techniques to elucidate the three-dimensional structure of the protein. These methods can provide insights into important features such as nucleotide binding sites, interaction surfaces with other subunits, and conformational changes associated with catalytic activity.

Site-directed mutagenesis offers a complementary approach for structure-function studies. By systematically altering specific amino acid residues and analyzing the effects on protein function, researchers can identify critical regions for catalytic activity, subunit interactions, or ion specificity. Particular attention should be given to residues that are conserved across ATP synthases from different species, as these often have essential functional roles.

Functional assays measuring ATP synthesis or hydrolysis under controlled conditions represent the third essential component of this research strategy. For ATP synthesis measurements, researchers can employ reconstituted proteoliposomes with established ion gradients, as demonstrated with other ATP synthases . The synthesis rates can be monitored over time using luciferase-based assays or other methods for ATP quantification.

How can Recombinant Shewanella baltica ATP synthase subunit alpha be used in comparative evolutionary studies?

Recombinant Shewanella baltica ATP synthase subunit alpha presents a valuable tool for comparative evolutionary studies of ATP synthases across diverse bacterial lineages. The ATP synthase complex demonstrates remarkable conservation in its fundamental mechanism while exhibiting specific adaptations across different organisms.

Sequence alignment studies comparing the Shewanella baltica atpA with homologs from other bacteria, archaea, and eukaryotes can reveal conserved functional domains and species-specific variations. Particular attention might be given to comparing Shewanella baltica ATP synthase with those from other bacteria with diverse respiratory capabilities or extreme environment adaptations .

Structural comparisons represent another valuable approach. By determining the structure of Shewanella baltica ATP synthase alpha subunit and comparing it with structures from other organisms, researchers can identify subtle adaptations that might correlate with specific environmental niches or metabolic strategies. This is particularly relevant given the unique respiratory versatility of Shewanella species, which can utilize a wide range of electron acceptors including solid substrates like electrodes .

Functional comparative studies can also be illuminating. By reconstituting ATP synthases with alpha subunits from different species, including Shewanella baltica, researchers can compare enzymatic parameters such as ion specificity, ATP synthesis rates, and threshold driving forces required for ATP synthesis .

What techniques can be used to investigate the interaction between ATP synthase subunit alpha and other components of the enzyme complex?

Investigating interactions between Recombinant Shewanella baltica ATP synthase subunit alpha and other components of the ATP synthase complex requires specialized techniques for protein-protein interaction analysis. These methods provide insights into the assembly, stability, and functional dynamics of the multisubunit enzyme complex.

Co-immunoprecipitation (Co-IP) represents a valuable approach for identifying protein-protein interactions. Antibodies against the alpha subunit, similar to those described for plant AtpA , can be used to precipitate the protein along with its interaction partners from solubilized ATP synthase complexes. The precipitated proteins can then be identified using mass spectrometry or Western blotting with subunit-specific antibodies.

Cross-linking mass spectrometry offers another powerful technique for mapping protein-protein interactions. By treating the assembled ATP synthase complex with chemical cross-linkers and subsequently analyzing the cross-linked peptides using mass spectrometry, researchers can identify specific contact points between the alpha subunit and neighboring proteins.

Surface plasmon resonance (SPR) or biolayer interferometry (BLI) can be employed for quantitative assessment of binding kinetics and affinities between the alpha subunit and other ATP synthase components. These techniques involve immobilizing one protein on a sensor surface and monitoring real-time binding of interaction partners in solution, yielding valuable quantitative data about association and dissociation rates.

How can this recombinant protein contribute to understanding bacterial energy metabolism at low driving forces?

Recombinant Shewanella baltica ATP synthase subunit alpha provides a valuable tool for investigating bacterial energy metabolism at low driving forces, an area of significant interest for understanding microorganisms that operate near the thermodynamic limits of energy conservation. This research direction is particularly relevant given the diverse respiratory capabilities of Shewanella species.

Threshold determination experiments represent a primary application in this context. By incorporating the recombinant alpha subunit into reconstituted ATP synthase complexes and measuring ATP synthesis rates at progressively lower ion gradients, researchers can determine the minimal driving force required for detectable ATP production. This threshold value provides crucial information about the energetic efficiency of the enzyme and can be compared with values established for other ATP synthases .

Recent research has demonstrated that some ATP synthases with unusual subunit compositions, such as those with V-type c subunits, can synthesize ATP at surprisingly low driving forces of 90 to 150 mV . Investigating whether Shewanella baltica ATP synthase shares this capability would provide valuable insights into the energetic adaptations of this bacterium to its ecological niche.

Comparative studies with ATP synthases from organisms known to operate at low driving forces can provide particularly valuable insights. By determining whether Shewanella baltica ATP synthase shares features with such specialized enzymes or employs different strategies for efficient energy conversion, researchers can expand our understanding of the diverse solutions to low-energy metabolism in bacterial systems .

What are the protocols for reconstituting functional ATP synthase complexes for in vitro ATP synthesis assays?

Reconstituting functional ATP synthase complexes for in vitro ATP synthesis assays requires careful consideration of protein components, lipid environment, and experimental conditions. While specific protocols for Shewanella baltica ATP synthase are not detailed in the available literature, principles can be adapted from successful approaches with other bacterial ATP synthases.

For protein preparation, researchers should purify all necessary ATP synthase subunits, including the recombinant alpha subunit, with attention to maintaining native conformations and functional properties. Depending on the experimental goals, this may involve either co-expression of multiple subunits or separate expression followed by in vitro assembly .

Proteoliposome preparation represents the core of the reconstitution process. Researchers should create liposomes with a defined lipid composition, considering both the lipid headgroups and acyl chain characteristics to mimic the native membrane environment of Shewanella baltica. The ATP synthase complex is then incorporated into these liposomes, typically through detergent-mediated reconstitution followed by detergent removal.

Creating ion gradients for driving ATP synthesis is the final critical step. For sodium-dependent ATP synthases, researchers can establish a sodium concentration gradient (e.g., 200 mM inside vs. 15 mM outside) combined with an electrical potential generated through a potassium diffusion potential using valinomycin . This approach can create a total driving force (ΔμNa+/F) of approximately 230 mV. For proton-dependent ATP synthases, similar principles apply with appropriate adjustments to establish the desired proton gradient.

How does the ion specificity of ATP synthases influence experimental design and results interpretation?

The ion specificity of ATP synthases significantly influences both experimental design and the interpretation of results in bioenergetic studies. Understanding whether an ATP synthase preferentially utilizes H+ or Na+ as coupling ions is fundamental to designing appropriate experiments and correctly interpreting the data obtained.

ATP synthases can utilize either protons (H+) or sodium ions (Na+) as the coupling ion for energy conversion. Some ATP synthases, such as the one from E. callanderi, demonstrate the ability to use either ΔpNa or Δψ as driving forces for ATP synthesis . In contrast, the H+-F1F0 ATP synthase of E. coli and the Na+-F1F0 ATP synthase of P. modestum require a combination of both ΔpH/ΔpNa and Δψ for effective ATP synthesis .

When designing experiments with the Shewanella baltica ATP synthase, researchers should implement controls that distinguish between H+ and Na+ driven activities. This includes experiments with specific ionophores that selectively dissipate either H+ gradients (using protonophores like TCS) or Na+ gradients (using Na+ ionophores like ETH2120) . The differential effects of these ionophores on ATP synthesis activity provide critical evidence for determining the coupling ion preference.

For quantitative assessments, researchers should establish the threshold values of different components of the electrochemical gradient (Δψ, ΔpH, ΔpNa) required for ATP synthesis. These threshold values provide important insights into the energetic requirements of the enzyme and allow meaningful comparisons with ATP synthases from other organisms .

What are the challenges and solutions in expressing and purifying functional recombinant ATP synthase subunits?

Expressing and purifying functional recombinant ATP synthase subunits presents several challenges due to the complex nature of these proteins and their native membrane environment. Understanding these challenges and implementing appropriate solutions is essential for obtaining high-quality recombinant proteins suitable for research applications.

Expression host selection represents a primary consideration. While the recombinant Shewanella baltica ATP synthase subunit alpha is produced in E. coli , researchers should consider the codon usage compatibility between the source and host organisms. For membrane-associated subunits, specialized expression systems designed for membrane proteins may be necessary to achieve proper folding and avoid toxicity to the host.

Protein solubility and folding present additional challenges, particularly for hydrophobic subunits or those that normally exist only in complex with other proteins. Fusion tags such as MBP (maltose-binding protein) or SUMO can enhance solubility, while chaperone co-expression may improve folding efficiency. For the alpha subunit specifically, which is part of the hydrophilic F1 sector, solubility issues may be less severe than for membrane-embedded components.

Purification strategy design must balance yield, purity, and functional preservation. The recombinant Shewanella baltica ATP synthase subunit alpha is reported to have a purity exceeding 85% as determined by SDS-PAGE , suggesting effective purification protocols are available. Affinity chromatography using engineered tags provides efficient initial capture, while subsequent polishing steps can achieve the desired purity level.

How can computational approaches complement experimental studies of ATP synthase subunit alpha?

Computational approaches provide powerful complementary tools for experimental studies of Recombinant Shewanella baltica ATP synthase subunit alpha, enabling analyses that may be challenging or impossible through laboratory methods alone. The integration of computational and experimental approaches creates a synergistic research strategy for understanding this complex protein.

Homology modeling and molecular dynamics simulations offer insights into the three-dimensional structure and dynamic behavior of the protein. Starting with the amino acid sequence of Shewanella baltica ATP synthase subunit alpha and using structures of homologous proteins as templates, researchers can generate structural models that predict important features such as nucleotide binding sites, interfaces with other subunits, and conformational flexibility.

Sequence-based evolutionary analyses provide another valuable computational approach. By comparing the sequence of Shewanella baltica ATP synthase subunit alpha with homologs from diverse organisms, researchers can identify conserved residues likely critical for function, detect signatures of positive selection suggesting adaptive evolution, and reconstruct the evolutionary history of specific protein features.

How can this recombinant protein be used to study the evolutionary adaptation of ATP synthases to different environments?

Recombinant Shewanella baltica ATP synthase subunit alpha serves as an excellent model for studying evolutionary adaptations of energy-transducing enzymes to different environmental conditions. The diverse ecological niches occupied by Shewanella species make their ATP synthases particularly interesting subjects for comparative evolutionary studies.

Comparative biochemical characterization represents a primary approach to investigating evolutionary adaptations. By determining the functional properties of the Shewanella baltica ATP synthase (including ion specificity, threshold driving force, temperature dependence, and pH optimum) and comparing these with ATP synthases from organisms in different environments, researchers can identify adaptive features that correlate with specific ecological niches .

Sequence analysis combined with structural modeling provides another powerful approach. By analyzing sequence variations in ATP synthase alpha subunits across bacteria from different environments and mapping these variations onto structural models, researchers can identify potentially adaptive changes in specific functional domains. Special attention should be given to regions involved in catalysis, subunit interactions, or conformational changes during the catalytic cycle.

Experimental validation of adaptive hypotheses can be conducted using site-directed mutagenesis of the recombinant protein. By introducing mutations that mimic the sequence variations observed in ATP synthases from organisms in different environments and measuring the functional consequences, researchers can directly test evolutionary hypotheses about the adaptive significance of specific amino acid changes.

What methodological approaches can investigate the rotational mechanics of ATP synthase using recombinant subunits?

Studying the rotational mechanics of ATP synthase represents one of the most fascinating aspects of this molecular machine's function. Advanced biophysical techniques have been developed to visualize and quantify the rotational movement that couples ion translocation to ATP synthesis, and these approaches can be adapted for studies incorporating recombinant Shewanella baltica ATP synthase subunit alpha.

Single-molecule fluorescence microscopy represents a powerful technique for directly observing rotation in ATP synthase. This approach typically involves attaching a fluorescent probe (such as a quantum dot or fluorescent bead) to the rotating portion of the enzyme (the c-ring or γ-subunit) and tracking its movement using high-resolution microscopy. The recombinant alpha subunit would be incorporated into the ATP synthase complex as part of the stationary portion (F1 sector), providing the structural framework against which rotation occurs.

Magnetic bead rotation assays offer an alternative approach for measuring rotational dynamics. In this technique, a magnetic bead is attached to the rotating component of ATP synthase, and its movement is tracked under an applied magnetic field. This method allows researchers to not only observe rotation but also to apply controlled torque to the system, providing insights into the mechanical properties of the enzyme.

Förster resonance energy transfer (FRET) between strategically placed fluorophores can be used to detect conformational changes associated with rotation. By incorporating fluorescent labels at specific positions within the alpha subunit and rotating components, researchers can monitor distance changes during the catalytic cycle, providing indirect evidence of rotational movement.