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

What is Recombinant Shewanella sediminis ATP synthase subunit alpha (atpA) and its role in cellular metabolism?

The ATP synthase subunit alpha (atpA) is one of the major catalytic components of the F1 sector of ATP synthase in S. sediminis. As part of the F1 complex (α3β3γδε), it forms a hexameric ring with alternating alpha and beta subunits that creates the catalytic sites essential for ATP synthesis. This subunit contributes to nucleotide binding and the conformational changes necessary for catalysis during energy production.

In S. sediminis, a psychrophilic bacterium isolated from marine sediments, ATP synthase plays a crucial role in energy metabolism by generating ATP through oxidative phosphorylation. This process is particularly significant in this organism's ability to thrive in cold, deep-sea environments where energy efficiency is paramount for survival .

How does the structure of S. sediminis ATP synthase alpha subunit compare with other bacterial homologs?

While specific structural information for S. sediminis atpA is not fully characterized in the available literature, comparative analysis with other bacterial ATP synthase alpha subunits indicates several expected features:

• A nucleotide-binding domain with a Rossmann fold typical of ATP-binding proteins

• Potential cold-adaptive structural modifications compared to mesophilic homologs

• Conserved catalytic residues common across bacterial ATP synthases

• Interfaces for interaction with beta subunits and other components of the F1 complex

Similar to other Shewanella proteins like the ATP synthase delta subunit (atpH), which has been more extensively characterized, the alpha subunit likely possesses adaptations that enable function at low temperatures while maintaining catalytic efficiency .

What are the recommended storage and handling conditions for recombinant S. sediminis atpA?

Based on established protocols for other recombinant S. sediminis ATP synthase components, the following handling conditions are recommended:

For optimal results, the purified protein should be aliquoted before freezing to avoid repeated freeze-thaw cycles that can compromise structural integrity and activity. The shelf life for liquid forms is typically 6 months at -20°C/-80°C, while lyophilized preparations may remain stable for up to 12 months .

What expression systems are most effective for producing recombinant S. sediminis atpA?

Heterologous expression in E. coli represents the most established system for producing recombinant S. sediminis ATP synthase components. This approach offers several advantages including:

• High yield of target protein

• Well-established genetic manipulation techniques

• Compatibility with various expression vectors and purification strategies

• Cost-effectiveness and scalability

For S. sediminis proteins, E. coli-based expression systems have demonstrated successful production of soluble, functional recombinant proteins as evidenced by the commercial availability of other ATP synthase components from this organism . Expression typically employs a T7 promoter system with IPTG induction and may incorporate affinity tags (His, GST, or MBP) to facilitate downstream purification.

What purification strategies yield the highest purity and activity for recombinant S. sediminis atpA?

A multi-step purification approach is recommended to achieve high purity (>85% by SDS-PAGE) while preserving the functional integrity of the protein :

• Initial Capture: Affinity chromatography using tags incorporated during cloning (His-tag is commonly employed)

• Intermediate Purification: Ion exchange chromatography to separate proteins based on charge differences

• Polishing Step: Size exclusion chromatography to remove aggregates and achieve final purity

Throughout the purification process, include protease inhibitors to prevent degradation and maintain cold conditions (4°C) to preserve protein structure and activity. Quality control at each step using SDS-PAGE analysis ensures tracking of purity progression. For psychrophilic proteins like those from S. sediminis, maintaining lower temperatures during purification may be particularly important for preserving native conformation and activity .

How can researchers validate the identity and functional integrity of purified recombinant S. sediminis atpA?

Validation requires both analytical and functional assessment approaches:

• Identity Confirmation:

  • SDS-PAGE for molecular weight verification

  • Western blot using specific antibodies or tag detection

  • Mass spectrometry for precise mass determination and peptide mapping

  • N-terminal sequencing to confirm the correct start of the protein

• Functional Assessment:

  • ATPase activity assays (measuring ATP hydrolysis)

  • Nucleotide binding studies using fluorescence or calorimetric methods

  • Interaction studies with other ATP synthase subunits

  • Temperature-dependent activity profiling to verify cold adaptation properties

• Structural Integrity:

  • Circular dichroism to assess secondary structure composition

  • Dynamic light scattering to evaluate homogeneity and oligomeric state

  • Thermal shift assays to determine stability and unfolding characteristics

A comprehensive validation approach combines these methods to ensure both the identity and functional capacity of the purified protein before proceeding with detailed biochemical or structural characterization .

What assays are most informative for characterizing the enzymatic activity of S. sediminis atpA?

Several complementary assays provide comprehensive insights into atpA function:

• ATP Hydrolysis Assays:

  • Colorimetric phosphate release detection (malachite green, molybdate)

  • Enzyme-coupled assays linking ATP hydrolysis to NADH oxidation

  • Real-time monitoring using pH-sensitive indicators

• Nucleotide Binding Studies:

  • Isothermal titration calorimetry (ITC) for binding energetics

  • Fluorescence-based assays using nucleotide analogs (TNP-ATP)

  • Surface plasmon resonance for binding kinetics

• Functional Reconstitution:

  • Assembly with other F1 subunits to assess complex formation

  • Proton pumping assays using reconstituted proteoliposomes

  • ATP synthesis measurements under appropriate conditions

For psychrophilic enzymes like those from S. sediminis, conducting these assays across a temperature range (4-25°C) can reveal cold-adaptive properties that distinguish them from mesophilic homologs .

How can researchers investigate temperature-dependent properties of S. sediminis atpA to understand cold adaptation?

As S. sediminis is a psychrophilic bacterium , its ATP synthase components likely exhibit specific cold-adaptive features that can be characterized through:

• Temperature-Activity Profiling:

  • Determine optimal temperature for activity

  • Measure activation energy using Arrhenius plots

  • Compare catalytic efficiency (kcat/Km) across temperature ranges

• Thermal Stability Analysis:

  • Differential scanning calorimetry to measure unfolding transitions

  • Temperature-dependent circular dichroism to monitor structural changes

  • Thermal shift assays using fluorescent dyes like SYPRO Orange

• Conformational Flexibility Studies:

  • Hydrogen-deuterium exchange mass spectrometry at different temperatures

  • Limited proteolysis to assess accessibility of cleavage sites

  • Molecular dynamics simulations to predict temperature effects on protein motion

• Comparative Analysis with Mesophilic Homologs:

  • Side-by-side activity and stability measurements

  • Sequence analysis to identify cold-adaptation signatures

  • Structural comparisons to identify flexibility-enhancing features

These approaches can reveal the molecular mechanisms that enable S. sediminis ATP synthase to function efficiently in cold marine sediment environments .

What approaches can assess the interaction between S. sediminis atpA and other ATP synthase subunits?

Understanding subunit interactions is crucial for elucidating ATP synthase assembly and function:

• Binding Affinity Measurements:

  • Co-immunoprecipitation with antibodies against atpA or other subunits

  • Surface plasmon resonance for real-time interaction kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

• Complex Formation Analysis:

  • Blue native PAGE to visualize intact complexes

  • Size exclusion chromatography to detect stable complexes

  • Analytical ultracentrifugation for stoichiometry determination

• Structural Approaches:

  • Cross-linking coupled with mass spectrometry to map interaction interfaces

  • Single-particle cryo-electron microscopy of assembled complexes

  • Homology modeling based on related ATP synthase structures

• Functional Reconstitution:

  • Step-wise assembly assays with purified components

  • Activity measurements of reconstituted complexes

  • Mutagenesis of predicted interface residues to validate interaction models

This multi-faceted approach can reveal how the S. sediminis atpA integrates into the functional ATP synthase complex and how these interactions might be adapted to the organism's psychrophilic lifestyle .

How does the genomic context of the atpA gene in S. sediminis provide insights into ATP synthase evolution and regulation?

Analysis of the genomic organization surrounding the atpA gene reveals important evolutionary and regulatory aspects:

• Operon Structure:

  • The atpA gene in Shewanella species typically resides in an operon with other ATP synthase genes

  • The order and arrangement of genes may differ from other bacterial groups

  • Promoter elements and regulatory regions can indicate conditions for expression

• Comparative Genomics:

  • Similar to the analysis performed for formate dehydrogenase genes in Shewanella species , ATP synthase genes show evolutionary patterns that reflect ecological adaptations

  • Gene duplication events may be identified, as observed with other energy metabolism genes

  • Horizontal gene transfer events can be detected through GC content and codon usage analysis

• Regulatory Networks:

  • Identification of transcription factor binding sites

  • Potential for coordinate regulation with other energy metabolism genes

  • Environmental response elements that reflect adaptation to marine sediment conditions

This genomic context analysis provides a framework for understanding how the atpA gene evolved within the Shewanella genus and how its expression might be regulated in response to environmental conditions .

How can structural biology approaches advance our understanding of S. sediminis atpA function and adaptation?

Structural biology techniques provide crucial insights into the molecular basis of atpA function:

• X-ray Crystallography:

  • High-resolution structures of isolated atpA or in complex with nucleotides

  • Identification of catalytic residues and binding pockets

  • Comparative analysis with mesophilic homologs to identify cold-adaptation features

• Cryo-Electron Microscopy:

  • Structure determination of the entire ATP synthase complex

  • Visualization of atpA in its native context within the F1 assembly

  • Capture of different conformational states during the catalytic cycle

• NMR Spectroscopy:

  • Solution structure of domains or the entire protein

  • Dynamics studies to assess flexibility at different temperatures

  • Binding interactions with nucleotides and other ligands

• Computational Approaches:

  • Homology modeling based on known ATP synthase structures

  • Molecular dynamics simulations to predict temperature-dependent behavior

  • Docking studies to investigate substrate and inhibitor interactions

These structural approaches, especially when conducted at temperatures relevant to S. sediminis' natural habitat, can reveal the molecular adaptations that enable ATP synthase function in cold marine sediments .

What is known about the role of post-translational modifications in regulating S. sediminis ATP synthase activity?

While specific information on post-translational modifications (PTMs) of S. sediminis ATP synthase is limited in the available literature, potential regulatory mechanisms can be inferred from related systems:

• Potential Modification Types:

  • Phosphorylation of serine, threonine, or tyrosine residues

  • Acetylation of lysine residues

  • Oxidation/reduction of cysteine residues

  • ADP-ribosylation as a regulatory mechanism

• Functional Consequences:

  • Modulation of catalytic activity in response to cellular energy status

  • Regulation of protein-protein interactions within the ATP synthase complex

  • Alteration of stability or half-life under different environmental conditions

  • Adaptation to varying oxygen levels, as Shewanella species are known for versatile respiratory capabilities

• Investigation Approaches:

  • Mass spectrometry-based proteomics to identify PTMs

  • Site-directed mutagenesis of potential modification sites

  • In vitro modification assays with relevant kinases or acetyltransferases

  • Antibodies specific for common PTMs to detect modifications in vivo

Understanding these regulatory mechanisms could provide insights into how S. sediminis fine-tunes ATP synthase activity in response to environmental changes in its marine sediment habitat .

How does the partial recombinant S. sediminis atpA compare functionally with the full-length protein?

The partial nature of the recombinant atpA has important implications for research applications:

• Structural Considerations:

  • The partial protein may lack certain domains needed for complete function

  • Key interaction interfaces with other subunits might be missing

  • Folding and stability could differ from the full-length protein

• Functional Implications:

  • Catalytic activity may be compromised if essential regions are absent

  • Nucleotide binding might be preserved if binding domains are intact

  • Regulatory mechanisms could be disrupted if regulatory domains are missing

• Research Utility:

  • Useful for domain-specific studies and antibody production

  • Valuable for structural studies of specific regions

  • May serve as a competitor in binding studies

• Experimental Design Considerations:

  • Clear definition of which regions are present and absent

  • Validation against full-length protein when possible

  • Careful interpretation of results in the context of known domain functions

Researchers should consider these limitations when designing experiments and interpreting results with the partial recombinant protein .

How does S. sediminis atpA compare with homologs from other Shewanella species found in different environments?

Comparative analysis across Shewanella species reveals adaptive patterns:

• Sequence Divergence Patterns:

  • Core catalytic regions typically show high conservation

  • Surface-exposed regions may exhibit greater variation

  • Similar evolutionary patterns may exist as observed with formate dehydrogenase genes across Shewanella species

• Habitat-Specific Adaptations:

  • Psychrophilic species (like S. sediminis) vs. mesophilic species

  • Deep-sea vs. shallow-water species

  • Marine vs. freshwater species

• Functional Implications:

  • Temperature optima correlating with natural habitat

  • Catalytic efficiency differences reflecting environmental energetics

  • Stability characteristics matching environmental challenges

• Evolutionary Insights:

  • Identification of positively selected residues indicating adaptive evolution

  • Potential gene duplication events as observed with other energy metabolism genes in Shewanella

  • Horizontal gene transfer contributions to adaptive capabilities

This comparative approach provides insights into how ATP synthase has evolved to support Shewanella species in colonizing diverse aquatic environments from deep-sea sediments to freshwater habitats .

What can we learn from comparing S. sediminis ATP synthase with those from extremophiles adapted to different environmental stresses?

Comparative analysis with extremophiles adapted to other conditions provides broader evolutionary insights:

• Adaptation Mechanisms Across Extremes:

  • Cold adaptation (psychrophiles) vs. heat adaptation (thermophiles)

  • Pressure adaptation strategies (piezophiles)

  • Halophilic adaptations for high salt environments

• Common Principles vs. Unique Solutions:

  • Convergent strategies in maintaining catalytic efficiency

  • Divergent approaches to structural stability

  • Balance between molecular flexibility and stability

• Structural Features Comparison:

  • Surface charge distribution patterns

  • Internal packing and hydrophobic core characteristics

  • Loop regions and flexibility-conferring elements

• Biotechnological Applications:

  • Identification of stability-enhancing features for protein engineering

  • Discovery of activity-preserving modifications for industrial applications

  • Design principles for creating enzymes functional under non-standard conditions

This comparative extremophile approach provides a broader context for understanding the fundamental principles of protein adaptation to environmental challenges and may inspire biomimetic approaches in biotechnology .

What strategies can address common challenges in expressing soluble S. sediminis atpA in heterologous systems?

Researchers may encounter several challenges when expressing this psychrophilic protein:

• Solubility Issues:

  • Lower expression temperature (16-20°C) to improve folding

  • Use solubility-enhancing fusion tags (SUMO, MBP, TrxA)

  • Co-express with molecular chaperones

  • Optimize induction conditions (reduced IPTG concentration, slower induction)

• Expression Level Optimization:

  • Codon optimization for the expression host

  • Try different promoter systems (T7, tac, araBAD)

  • Screen multiple E. coli strains (BL21(DE3), Rosetta, Arctic Express)

  • Evaluate different media compositions

• Preserving Activity:

  • Include stabilizing agents in lysis buffer

  • Minimize time between cell disruption and purification

  • Add potential cofactors or ligands during extraction

  • Maintain cold chain throughout processing

• Structured Troubleshooting Approach:

  • Systematically vary one parameter at a time

  • Perform small-scale expression tests before scaling up

  • Analyze both soluble and insoluble fractions

  • Consider cell-free expression systems for difficult proteins

These strategies can significantly improve the yield and quality of recombinant S. sediminis atpA protein for subsequent structural and functional studies .

How can researchers address stability issues with purified S. sediminis atpA during storage and experimental procedures?

Maintaining stability of this psychrophilic protein requires specific considerations:

• Buffer Optimization:

  • Screen different pH values (typically 7.0-8.0)

  • Test various salt concentrations (100-300 mM NaCl)

  • Include stabilizing agents (5-20% glycerol, 1-5 mM DTT)

  • Add specific ions that might enhance stability (Mg²⁺, K⁺)

• Storage Conditions:

  • Store at -80°C for long-term preservation

  • Aliquot to avoid repeated freeze-thaw cycles

  • Consider flash-freezing in liquid nitrogen

  • Evaluate lyophilization for extended shelf life (up to 12 months)

• Handling During Experiments:

  • Maintain cold temperatures during all manipulations

  • Minimize exposure to air/oxidation

  • Use low-binding tubes to prevent surface adsorption

  • Add carrier proteins (BSA) for very dilute solutions

• Stability Assessment Methods:

  • Regular activity assays to monitor functional integrity

  • Size exclusion chromatography to detect aggregation

  • Dynamic light scattering to assess homogeneity

  • Thermal shift assays to evaluate stability under different conditions

These approaches can significantly extend the useful lifetime of purified protein and ensure consistent results across experiments .

What are effective approaches for troubleshooting activity assays with recombinant S. sediminis atpA?

When activity assays yield unexpected results, systematic troubleshooting can identify and resolve issues:

• Common Problems and Solutions:

  Problem	Potential Solutions
  Low or no activity	- Verify protein integrity by SDS-PAGE - Check for inhibitory contaminants - Ensure cofactors (Mg²⁺) are present - Optimize assay temperature (consider cold adaptation)
  Inconsistent results	- Standardize protein concentration determination - Control reaction temperature precisely - Prepare fresh substrates and reagents - Use internal controls
  Interfering factors	- Examine buffer components for interference - Dialyze protein thoroughly - Test alternative assay methods - Consider enzyme-coupled assays
  Rapid activity loss	- Check for proteolytic degradation - Add protease inhibitors - Optimize protein stability conditions - Prepare fresh enzyme before assays

• Optimization Approaches:

  • Perform temperature optimization (especially important for psychrophilic enzymes)

  • Determine optimal pH through activity profiles

  • Titrate cofactor concentrations

  • Evaluate buffer components systematically

• Alternative Assay Methods:

  • If one assay method fails, try complementary approaches

  • Consider direct (product formation) and indirect (coupled) assays

  • Investigate label-free detection methods

  • Use sensitive instrumentation for low-activity detection

This structured approach to troubleshooting can resolve most activity assay issues and lead to reliable, reproducible results .

What emerging technologies could advance our understanding of S. sediminis ATP synthase structure and function?

Several cutting-edge technologies offer promising avenues for future research:

• Advanced Structural Techniques:

  • Cryo-electron tomography for studying ATP synthase in native membrane environments

  • Micro-electron diffraction for structural analysis from nanocrystals

  • Single-particle cryo-EM with improved resolution for capturing catalytic states

  • Time-resolved structural methods to visualize conformational dynamics

• Functional Analysis Innovations:

  • Single-molecule techniques to observe individual catalytic events

  • High-sensitivity nanocalorimetry for thermodynamic profiling

  • Improved biosensors for real-time activity monitoring

  • Fluorescence-based approaches to track conformational changes

• Genetic and Genomic Approaches:

  • CRISPR-Cas9 genome editing in Shewanella species

  • Deep mutational scanning to comprehensively map structure-function relationships

  • Improved heterologous expression systems for challenging proteins

  • Synthetic biology approaches for reconstructing minimal ATP synthase systems

• Computational Methods:

  • Enhanced sampling molecular dynamics simulations for cold adaptation studies

  • Machine learning approaches to predict function from sequence

  • Quantum mechanics/molecular mechanics for catalytic mechanism studies

  • Systems biology modeling of ATP synthase in cellular context

These technologies can provide unprecedented insights into how S. sediminis ATP synthase functions in its psychrophilic marine sediment environment .

How might research on S. sediminis ATP synthase contribute to understanding microbial adaptation to deep-sea environments?

S. sediminis ATP synthase research has broader implications for deep-sea microbial ecology:

• Bioenergetic Adaptations:

  • Mechanisms for efficient energy conservation at low temperatures

  • Adaptations to hydrostatic pressure effects on protein function

  • Bioenergetic trade-offs in extreme environments

  • Integration with other uniquely adapted metabolic pathways

• Evolutionary Considerations:

  • Selection pressures shaping ATP synthase evolution in deep-sea niches

  • Convergent adaptations across phylogenetically distant deep-sea microbes

  • Role of horizontal gene transfer in adaptation to deep-sea environments

  • Molecular clock analyses to date adaptations to deep-sea conditions

• Ecological Implications:

  • Contribution to niche differentiation among deep-sea microbes

  • Role in enabling colonization of specific marine sediment microhabitats

  • Connections to biogeochemical cycling in deep-sea environments

  • Potential interactions with other organisms through energy-related adaptations

• Climate Change Relevance:

  • Potential responses to changing ocean temperatures

  • Adaptability to shifting ocean chemistry

  • Implications for deep-sea ecosystem functioning under changing conditions

  • Predictive models for microbial community shifts based on bioenergetic adaptations

This research connects molecular adaptations to ecosystem-level processes in deep-sea environments .

What potential biotechnological applications might emerge from studies of S. sediminis ATP synthase?

The unique properties of this psychrophilic ATP synthase could lead to several applications:

• Enzyme Technology:

  • Cold-active enzymes for industrial processes at reduced temperatures

  • Energy-efficient biocatalysts for biotransformations

  • Templates for engineering stable and active proteins

  • Novel bioenergetic components for synthetic biology

• Biosensing Applications:

  • ATP detection systems functional at low temperatures

  • Environmental monitoring tools for cold environments

  • Highly sensitive energy metabolite detection platforms

  • Integration into biohybrid devices

• Biomedicine and Pharmacology:

  • Targets for developing antibacterial compounds with novel mechanisms

  • Model systems for studying human ATP synthase-related disorders

  • Templates for designing inhibitors of pathogenic bacteria

  • Tools for mitochondrial research at physiological temperatures

• Bioenergetic Technologies:

  • Bio-inspired energy conversion systems

  • Components for artificial photosynthesis

  • Nanoscale rotary motors for molecular machines

  • Models for developing energy-efficient nanoscale devices

These applications leverage the unique adaptations of S. sediminis ATP synthase to create technologies that operate efficiently under conditions where conventional enzymes may perform poorly .