Recombinant Desulfotalea psychrophila ATP synthase subunit c (atpE)

What is the structural composition of Desulfotalea psychrophila ATP synthase subunit c?

Desulfotalea psychrophila ATP synthase subunit c (atpE) is a small membrane protein consisting of 83 amino acids with the sequence: MEGNIQLALICVGAALSIGLAGLGAGIGIGSVGQGACMGLARNPEVQPKLMVFMILGMALAESIAIYGLVISLILLYANPLLG. The protein forms part of the F0 sector of ATP synthase, specifically within the c-ring structure that facilitates ion translocation across the membrane. Recombinant versions typically include an N-terminal His-tag to facilitate purification while maintaining structural integrity. The protein's hydrophobic nature reflects its membrane-embedded position within the ATP synthase complex .

How does Desulfotalea psychrophila ATP synthase subunit c compare with homologs from mesophilic organisms?

Desulfotalea psychrophila is a psychrophilic (cold-loving) sulfate-reducing bacterium, and its ATP synthase components, including subunit c, have evolved to function efficiently at low temperatures. Comparative sequence analysis reveals specific adaptations that distinguish it from mesophilic homologs. These adaptations typically include:

• Increased flexibility in structural elements

• Modified hydrophobic interactions

• Altered ion-binding sites optimized for function at lower temperatures

When designing experiments using this protein, researchers should consider these psychrophilic adaptations, particularly when comparing functional characteristics with mesophilic counterparts, as temperature optimization will significantly impact experimental outcomes .

What expression systems are most effective for producing recombinant Desulfotalea psychrophila ATP synthase subunit c?

E. coli expression systems have proven most effective for recombinant production of Desulfotalea psychrophila ATP synthase subunit c. Specifically, E. coli strains optimized for membrane protein expression (such as C41(DE3) or C43(DE3)) yield better results than standard laboratory strains. The protein is typically expressed with an N-terminal His-tag to facilitate purification.

Methodology recommendations:

• Use a vector with a moderate-strength promoter to avoid toxic overexpression

• Express at lower temperatures (16-20°C) to improve folding

• Include membrane-stabilizing additives in the growth medium

• Consider codon optimization for E. coli expression if yields are low

The recombinant protein can be obtained with greater than 90% purity using nickel affinity chromatography followed by size exclusion chromatography .

How can Desulfotalea psychrophila ATP synthase subunit c be reconstituted into liposomes for functional studies?

Functional reconstitution of Desulfotalea psychrophila ATP synthase subunit c into liposomes requires careful attention to membrane composition and reconstitution conditions. The methodology involves:

• Preparation of purified protein in a detergent-solubilized state

• Liposome preparation using a mixture of phospholipids (typically DOPC/DOPE/DOPG at 7:2:1 ratio)

• Gradual detergent removal using either dialysis or Bio-Beads

• Verification of successful incorporation using freeze-fracture electron microscopy

For optimal functional reconstitution, researchers should maintain a protein-to-lipid ratio of approximately 1:100 (w/w). The resulting proteoliposomes can be used for ion transport studies and ATP synthesis assays. Success of reconstitution can be verified by measuring protein orientation using protease accessibility assays .

What methods are most effective for measuring ATP synthesis activity of reconstituted Desulfotalea psychrophila ATP synthase?

ATP synthesis activity of reconstituted Desulfotalea psychrophila ATP synthase can be measured using a continuous luciferase-based assay. The methodology involves:

• Preparation of proteoliposomes containing reconstituted ATP synthase

• Generation of ion gradients (either Na+ or H+ depending on specificity)

• Addition of ADP and phosphate

• Real-time monitoring of ATP production

Protocol Details:

• Perform measurements at 37°C in a luminometer using a white flat-bottomed 96-well microtiter plate

• Mix 275 μl of proteoliposomes with 20 μL ATP Bioluminescence Assay Kit

• Record baseline for 3 minutes

• Initiate ATP synthesis by adding 0.5 mM ADP and 2 μM valinomycin

• Conduct measurements in triplicates from three independent experiments for statistical validity

The assay can be modified to investigate the influence of different parameters (Δψ, ΔpNa, ion concentrations) on ATP synthesis rates .

How does ionic specificity affect the function of Desulfotalea psychrophila ATP synthase subunit c?

Ionic specificity is a critical aspect of ATP synthase function, with some bacterial ATP synthases being Na+-specific rather than H+-dependent. While specific ionic preference data for Desulfotalea psychrophila ATP synthase is limited in the provided search results, research on other bacterial ATP synthases provides a methodological framework for investigation:

• Compare ATP synthesis rates at varying Na+ and H+ concentrations

• Analyze the effect of ion-competing inhibitors (e.g., DCCD) on activity

• Perform site-directed mutagenesis of key binding residues

Similar to E. callanderi ATP synthase, which shows Na+-dependence with half-maximal activity at 0.57 mM Na+, the Desulfotalea psychrophila enzyme may exhibit specific ion preferences adapted to its environmental niche. Researchers should test ATP synthesis activity with various ion concentration gradients while maintaining constant membrane potential (Δψ) to determine ionic specificity .

What role does the Desulfotalea psychrophila ATP synthase play in psychrophilic adaptation?

Desulfotalea psychrophila has evolved to thrive in cold environments, and its ATP synthase represents a key adaptation enabling energy production at low temperatures. Research approaches to investigate psychrophilic adaptations include:

• Comparative activity assays at different temperatures (0-37°C)

• Thermal stability studies using differential scanning calorimetry

• Molecular dynamics simulations to identify flexible regions

The c-ring of ATP synthase, composed of multiple copies of subunit c, must maintain both structural integrity and rotational flexibility at low temperatures. Analyzing how sequence variations in Desulfotalea psychrophila atpE contribute to cold adaptation provides insights into fundamental bioenergetic adaptation mechanisms. Experimental data suggests psychrophilic ATP synthases typically show higher catalytic rates at lower temperatures compared to mesophilic homologs, with structural modifications that enhance flexibility while maintaining necessary stability .

How does the c-ring stoichiometry of Desulfotalea psychrophila ATP synthase compare with other bacterial ATP synthases?

• Atomic force microscopy of isolated c-rings

• Mass determination using non-denaturing mass spectrometry

• Cross-linking studies followed by SDS-PAGE analysis

• Cryo-electron microscopy structural analysis

C-ring stoichiometry typically ranges from 8-15 subunits across different species, with psychrophilic organisms often showing adaptations in this parameter to optimize energy conversion at lower temperatures. Determining the precise stoichiometry would reveal important insights into bioenergetic adaptations of Desulfotalea psychrophila to cold environments .

What are the key challenges in maintaining stability of purified Desulfotalea psychrophila ATP synthase subunit c?

The hydrophobic nature of ATP synthase subunit c presents significant challenges for maintaining stability during purification and storage. Key considerations include:

Storage Recommendations:

• Store the lyophilized powder at -20°C/-80°C upon receipt

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles

• Add 5-50% glycerol (final concentration) for long-term storage

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

Stability Challenges and Solutions:

• Aggregation: Use stabilizing detergents (DDM or LMNG) at concentrations above CMC

• Oxidation: Include reducing agents such as DTT or TCEP

• Proteolytic degradation: Add protease inhibitors during handling

• Temperature sensitivity: Maintain cold chain throughout purification

Avoiding repeated freeze-thaw cycles is particularly important as membrane proteins are susceptible to denaturation during this process. For working stocks, store aliquots at 4°C for up to one week rather than repeatedly freezing and thawing samples .

How can researchers accurately determine the orientation of Desulfotalea psychrophila ATP synthase when reconstituted into liposomes?

Determining the orientation of ATP synthase in proteoliposomes is crucial for functional studies, as only correctly oriented complexes will contribute to measurable activity. Methodological approaches include:

• Protease Accessibility Assays: Expose proteoliposomes to proteases that selectively cleave exposed domains, followed by SDS-PAGE analysis

• Antibody Binding: Use antibodies against domains expected to be on either the inside or outside

• Fluorescence Quenching: Label specific residues with fluorescent probes and measure accessibility to membrane-impermeable quenchers

• Activity Measurements: Compare ATP synthesis versus hydrolysis activities, which depend on orientation

Typically, reconstitution procedures result in mixed orientations. Researchers can enrich for desired orientations by:

• Adjusting lipid composition

• Modifying reconstitution pH

• Using directed reconstitution methods with pre-formed liposomes

For experimental design, it's important to quantify the proportion of correctly oriented complexes to accurately interpret activity measurements .

How does Desulfotalea psychrophila ATP synthase subunit c interact with the sulfate reduction pathway?

Desulfotalea psychrophila is a sulfate-reducing bacterium, and understanding the integration of its ATP synthase with the sulfate reduction pathway provides insights into bioenergetic coupling. Research approaches include:

• Gene proximity analysis within the genome

• Co-expression studies under different sulfate availability conditions

• Protein-protein interaction studies using cross-linking or co-immunoprecipitation

Similar to other sulfate-reducing bacteria, Desulfotalea psychrophila shows proximity between ATP synthase genes and sulfate reduction pathway genes. For example, the qmo genes are located near the aps genes in multiple sulfate-reducing bacteria including Desulfotalea psychrophila. This genomic organization suggests functional coupling between ATP synthesis and sulfate reduction pathways .

Researchers investigating this interaction should consider:

• Measuring ATP synthesis rates under different sulfate reduction conditions

• Analyzing membrane potential generation during sulfate reduction

• Examining how electron transport components couple to ATP synthase activity

What experimental approaches can determine if Desulfotalea psychrophila ATP synthase is specifically involved in cold adaptation mechanisms?

To determine the specific role of ATP synthase in cold adaptation, researchers can employ several experimental approaches:

• Comparative Growth Studies:

  • Compare growth rates of wild-type and ATP synthase mutants at different temperatures

  • Analyze ATP production capacity across a temperature range (0-30°C)

• Heterologous Expression:

  • Express Desulfotalea psychrophila ATP synthase genes in mesophilic hosts

  • Test whether this confers enhanced cold tolerance or energy production at low temperatures

• Structural Analysis:

  • Perform cryo-EM or X-ray crystallography at different temperatures

  • Identify flexible regions that may contribute to cold adaptation

• Mutagenesis Studies:

  • Create chimeric ATP synthases with components from mesophilic organisms

  • Identify which domains are critical for cold adaptation

These approaches collectively provide insights into whether and how the ATP synthase complex specifically contributes to Desulfotalea psychrophila's ability to thrive in cold environments .

What spectroscopic methods are most effective for studying the structure-function relationship of Desulfotalea psychrophila ATP synthase subunit c?

Several spectroscopic techniques are particularly valuable for analyzing structure-function relationships in ATP synthase subunit c:

• Circular Dichroism (CD) Spectroscopy:

  • Provides information about secondary structure content (α-helical content)

  • Can monitor thermal stability and unfolding transitions

  • Useful for comparing wild-type and mutant proteins

• Fourier Transform Infrared (FTIR) Spectroscopy:

  • Offers detailed information about protein secondary structure in membrane environments

  • Can detect subtle changes in hydrogen bonding and protonation states

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Enables atomic-level structural analysis of isotope-labeled proteins

  • Can provide dynamics information through relaxation measurements

  • Particularly useful for studying ion-binding sites

• Fluorescence Spectroscopy:

  • When combined with site-directed incorporation of fluorescent amino acids or labels

  • Enables monitoring of conformational changes during function

  • Can be used to study protein-lipid interactions

For ATP synthase subunit c specifically, solid-state NMR has proven particularly valuable due to its ability to analyze membrane proteins in their native-like lipid environments .

How can site-directed mutagenesis be effectively applied to study ion-binding sites in Desulfotalea psychrophila ATP synthase subunit c?

Site-directed mutagenesis is a powerful approach for investigating ion-binding sites in ATP synthase subunit c. An effective experimental design includes:

• Target Selection:

  • Identify conserved residues in ion-binding sites based on sequence alignments

  • Focus on acidic residues (Asp, Glu) typically involved in ion coordination

  • Consider residues unique to psychrophilic species

• Mutation Strategy:

  • Conservative substitutions (e.g., Asp to Asn) to maintain structure but alter charge

  • Non-conservative substitutions to dramatically alter binding properties

  • Alanine scanning of suspected binding regions

• Functional Characterization:

  • Reconstitute mutant proteins into liposomes

  • Measure ATP synthesis at varying ion concentrations

  • Determine apparent Km values for different ions

  • Analyze inhibitor sensitivity (e.g., DCCD binding)

• Structural Validation:

  • Confirm mutant protein folding using CD spectroscopy

  • Perform thermal stability analysis to ensure mutations don't destabilize the protein

For analyzing results, construct binding curves for each mutant and determine whether the mutations affect affinity (Km) or maximal activity (Vmax), which provides mechanistic insights into ion binding and translocation .

How should researchers interpret ATP synthesis data from reconstituted Desulfotalea psychrophila ATP synthase experiments?

Proper interpretation of ATP synthesis data requires careful consideration of multiple factors:

• Control Experiments:

  • Include negative controls (proteoliposomes without protein or with denatured protein)

  • Use specific inhibitors (e.g., DCCD) to verify ATP synthase-specific activity

  • Perform measurements with and without ion gradients

• Data Normalization:

  • Normalize activity to protein amount to enable comparison between preparations

  • Account for protein orientation in proteoliposomes (typically 50-60% right-side-out)

  • Consider efficiency of reconstitution between different preparations

• Kinetic Analysis:

  • Plot initial rates rather than endpoint measurements

  • Construct Michaelis-Menten curves for substrate (ADP, Pi) dependence

  • Analyze ion concentration dependencies

• Thermodynamic Considerations:

  • Calculate theoretical ATP yield based on applied ion gradients

  • Compare experimental yields to theoretical maximums

  • Consider effects of membrane potential (Δψ) separately from ion gradients (ΔpH or ΔpNa)

When reporting results, present both raw data and normalized values, with clear explanation of normalization procedures. Statistical analysis should include triplicate measurements from at least three independent experiments, as this is standard practice for ATP synthesis measurements .

What are the critical factors affecting reproducibility in ATP synthase functional assays?

Several critical factors impact reproducibility in ATP synthase functional assays:

Table 1: Critical Factors Affecting Reproducibility in ATP Synthase Assays

To ensure reproducibility, researchers should:

• Develop detailed standard operating procedures

• Include internal standards in each experiment

• Report all experimental parameters in publications

• Validate critical findings across multiple protein preparations

What emerging technologies might advance our understanding of Desulfotalea psychrophila ATP synthase subunit c function?

Several emerging technologies show promise for advancing our understanding of ATP synthase structure and function:

• Cryo-Electron Tomography:

  • Enables visualization of ATP synthase in situ within native membranes

  • Provides insights into supramolecular organization and interactions with other complexes

• Single-Molecule FRET:

  • Allows real-time monitoring of conformational changes during catalysis

  • Can detect intermediates not observable in ensemble measurements

• Nanodiscs and Styrene-Maleic Acid Lipid Particles (SMALPs):

  • Provide more native-like membrane environments than detergent solubilization

  • Enable study of lipid-protein interactions critical for function

• Microfluidic Devices:

  • Allow precise control of environmental conditions

  • Enable high-throughput screening of functional properties

• Computational Approaches:

  • Machine learning for structure prediction

  • Molecular dynamics simulations at extended timescales

  • Quantum mechanical/molecular mechanical (QM/MM) approaches for investigating ion binding

These technologies, when applied to Desulfotalea psychrophila ATP synthase, could reveal unique adaptations enabling function at low temperatures and provide deeper insights into the evolution of bioenergetic systems .

How might research on Desulfotalea psychrophila ATP synthase inform synthetic biology applications?

Research on Desulfotalea psychrophila ATP synthase has several potential applications in synthetic biology:

• Creating Cold-Active Biocatalysts:

  • Engineering mesophilic organisms with psychrophilic ATP synthase components

  • Developing energy-efficient biotechnological processes at low temperatures

• Designing Minimal Cells:

  • Incorporating simplified or optimized ATP synthase variants

  • Creating energy-generating modules for synthetic cells

• Nanomachine Development:

  • Using ATP synthase as a template for designing synthetic molecular motors

  • Adapting cold-adapted features for improved function in artificial systems

• Biosensors:

  • Developing ATP synthase-based sensors for detecting ion gradients or ATP production

  • Creating environmental monitoring tools for extreme conditions

To pursue these applications, researchers should:

• Characterize the minimal structural elements required for function

• Identify specific adaptations enabling cold activity

• Develop methods for incorporating ATP synthase into non-native membranes

• Explore hybrid systems combining components from different organisms