Recombinant Dictyoglomus turgidum ATP synthase subunit c (atpE)

What is Dictyoglomus turgidum and why is its ATP synthase subunit c of interest to researchers?

Dictyoglomus turgidum is a chemoorganotrophic, extremely thermophilic, Gram-negative, strictly anaerobic bacterium isolated from a hot spring in the Uzon Caldera, eastern Kamchatka, Russia. This organism has gained significant attention in biochemical research due to its ability to thrive at extremely high temperatures (optimally at 72°C) despite having an anomalously low G+C content of 33.96% .

D. turgidum and D. thermophilum together constitute the Dictyoglomi phylum, a distinct bacterial lineage with unique evolutionary characteristics. What makes D. turgidum particularly interesting is its metabolic versatility - it can grow on a wide range of polysaccharide substrates including starch, cellulose, pectin, carboxymethylcellulose, lignin, and humic acids, although it cannot utilize pentose sugars such as xylose and arabinose .

The ATP synthase subunit c (atpE) from this organism is of particular interest because:

• It functions under extreme temperature conditions

• It may contain unique structural adaptations for thermostability

• Its properties could inform the design of heat-resistant biocatalysts

• It represents an evolutionarily distinct ATP synthase system

For researchers investigating membrane proteins, energy conversion mechanisms, or thermostable enzymes, the ATP synthase components from D. turgidum offer valuable model systems for understanding protein stability and function under extreme conditions.

What are the optimal growth conditions for culturing D. turgidum for experimental work?

Successful cultivation of D. turgidum requires carefully controlled anaerobic conditions reflecting its natural hot spring habitat. Based on established protocols for thermophilic anaerobes and the specific requirements of D. turgidum, the following conditions are recommended:

Temperature: Optimal growth occurs at 72°C, with a growth range of 50-80°C .

pH: Maintain medium at approximately pH 7.0 for optimal growth.

Atmosphere: Strictly anaerobic conditions must be maintained, typically using an anaerobic chamber or sealed vessels with reducing agents.

Basic Medium Composition:

• Base medium containing essential salts (similar to TRM medium)

• Nitrogen source (often ammonium sulfate at 0.5 g/L)

• Phosphate buffer system

• Trace minerals including magnesium, potassium, and calcium

• Reducing agents (typically sodium sulfide)

• Carbon sources: Complex substrates such as yeast extract and tryptone support robust growth

Carbon Sources: D. turgidum can utilize:

• Starch

• Cellulose

• Pectin

• Carboxymethylcellulose

• Complex protein sources (yeast extract, peptone, tryptone)

Growth Monitoring: Cell density can be monitored using spectrophotometric methods, protein concentration assays, or direct microscopic counting.

For researchers planning to express recombinant ATP synthase components, robust growth is essential to obtain sufficient biomass for subsequent protein purification steps.

What are the optimal conditions for heterologous expression of D. turgidum ATP synthase subunit c (atpE)?

Heterologous expression of thermophilic membrane proteins like ATP synthase subunit c presents significant challenges. Based on protocols established for similar proteins from thermophilic organisms, the following methodological approach is recommended:

Expression Systems:

• E. coli BL21(DE3): Most commonly used for initial expression trials

  • Advantages: Well-established protocols, rapid growth

  • Disadvantages: May form inclusion bodies with thermophilic proteins

• E. coli C41(DE3) or C43(DE3): Engineered for membrane protein expression

  • Advantages: Reduced toxicity from membrane protein overexpression

  • Disadvantages: Lower yields than inclusion body formation

• Cell-free expression systems: Alternative for difficult-to-express proteins

  • Advantages: Avoids toxicity issues, allows addition of detergents/lipids

  • Disadvantages: Higher cost, potentially lower yield

Expression Vector Considerations:

• Low-copy number vectors often perform better for membrane proteins

• Fusion tags (His6, MBP, SUMO) can improve solubility

• Inducible promoters (T7, rhamnose) allow controlled expression

Protocol Optimization Table:

Extraction and Solubilization:
After expression, a protocol similar to that used for other His-tagged proteins can be employed , with modifications specific to membrane proteins:

• Cell lysis using detergent-based methods (Cellytic B reagent)

• Membrane fraction isolation through differential centrifugation

• Selective solubilization using mild detergents (DDM, LDAO)

• Purification using affinity chromatography under detergent-stabilized conditions

As observed with other D. turgidum proteins, a rhamnose-inducible promoter system with N-terminal histidine tags has proven effective for various enzymes from this organism .

How does the ATP synthase subunit c (atpE) from D. turgidum compare structurally with homologs from other thermophiles?

ATP synthase subunit c from thermophilic organisms typically displays specific adaptations that contribute to protein stability at high temperatures. While specific structural information for D. turgidum ATP synthase subunit c is limited, comparative analysis with related thermophiles suggests the following structural characteristics:

Key Structural Features:

• Increased Hydrophobicity: Enhanced core packing through additional hydrophobic interactions

• Ion-Pair Networks: Extensive salt bridge networks on protein surfaces

• Reduced Flexibility: Fewer glycine residues in loop regions

• Disulfide Bonds: Potential additional covalent stabilization

• Proline Residues: Strategically positioned to reduce entropy of unfolding

Comparative Sequence Analysis:
Based on patterns observed in ATP synthase subunit c from related thermophiles, D. turgidum atpE likely shows:

• Higher alanine and leucine content than mesophilic homologs

• Fewer thermolabile residues (Asn, Gln, Met, Cys) in exposed positions

• More charged residues forming stabilizing networks

Predicted Transmembrane Topology:
The canonical ATP synthase subunit c forms a hairpin structure with two transmembrane helices connected by a polar loop. In thermophiles, this basic structure is preserved with specific stabilizing modifications to withstand high temperatures.

The unusually low G+C content of D. turgidum (33.96%) creates an interesting paradox regarding protein thermostability, suggesting that unusual mechanisms for thermal adaptation may be present in this organism's proteins.

What enzymatic assays are suitable for characterizing recombinant D. turgidum ATP synthase activity?

Functional characterization of recombinant ATP synthase requires assays that can:

• Measure ATP hydrolysis (ATPase activity)

• Assess proton translocation

• Determine the coupling between these two processes

Methodological Approaches:

ATPase Activity Assays:

• Coupled Enzyme Assay: Links ATP hydrolysis to NADH oxidation via pyruvate kinase and lactate dehydrogenase

• Malachite Green Assay: Colorimetric detection of released phosphate

• Luciferase-Based Assay: Measures ATP depletion directly

Proton Translocation Assays:

• pH Indicator Dyes: ACMA (9-amino-6-chloro-2-methoxyacridine) fluorescence quenching

• pH Electrode Measurements: Direct monitoring of pH changes

• Membrane Potential Probes: Voltage-sensitive dyes (e.g., oxonol VI)

Thermostability Assessment:

• Differential Scanning Calorimetry (DSC): Determines thermal transition points

• Circular Dichroism (CD): Monitors secondary structure changes with temperature

• Activity Persistence Assay: Measures activity retention after thermal challenge

Reconstitution Systems:

• Proteoliposomes: Phospholipid vesicles with incorporated ATP synthase

• Nanodiscs: Membrane protein stabilization in disc-shaped phospholipid bilayers

Temperature Considerations:
When working with enzymes from D. turgidum, assays should be performed at physiologically relevant temperatures (optimal growth temperature is 72°C) . This requires:

• Temperature-stable buffers (PIPES, HEPES)

• Preheating of reaction components

• Temperature-controlled spectrophotometers or plate readers

A key challenge in these assays is maintaining stability of coupling enzymes or indicators at the high temperatures required for optimal D. turgidum enzyme function.

What approaches can be used to study structure-function relationships in D. turgidum ATP synthase subunit c?

Understanding structure-function relationships in ATP synthase subunit c requires a multi-faceted approach combining genetic, biochemical, and biophysical techniques:

1. Site-Directed Mutagenesis:
Target residues for mutation based on:

• Sequence conservation analysis across thermophiles

• Homology modeling predictions

• Known functional sites (e.g., the conserved carboxylate in the ion-binding site)

A systematic mutagenesis approach might involve:

• Charge neutralization of key residues

• Conservative vs. non-conservative substitutions

• Introduction/removal of potential stabilizing interactions

Chimeric Protein Construction:

• Exchange domains between thermophilic and mesophilic ATP synthase c-subunits

• Analyze which regions confer thermostability or altered function

Biophysical Characterization:

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Maps protein dynamics and solvent accessibility

• NMR Spectroscopy: For structural analysis of specific residues

• Cryo-EM: For structural determination of the entire ATP synthase complex

Computational Methods:

• Molecular Dynamics Simulations: Probe dynamics at high temperatures

• Protein Energy Landscape Exploration: Identify stabilizing interactions

• Electrostatic Surface Mapping: Analyze charged residue networks

5. Functional Assessment:
For each variant created, assess:

• Protein expression and membrane integration

• Thermostability profile

• ATP hydrolysis activity

• Proton translocation efficiency

• Assembly with other ATP synthase subunits

Experimental Design Table:

This multi-technique approach allows researchers to correlate specific structural features with thermostability, ion specificity, and catalytic efficiency of the ATP synthase subunit c.

What expression systems and purification strategies are most effective for obtaining functional D. turgidum ATP synthase components?

Obtaining functional thermophilic membrane proteins presents unique challenges requiring specialized expression and purification approaches:

Expression Systems Comparison:

Bacterial Expression Optimization:
For D. turgidum proteins, protocols similar to those used for DNA polymerase I expression have proven successful :

• Cloning into rhamnose-inducible vector systems

• Expression in standard E. coli strains with N-terminal histidine tags

• Induction at moderate temperatures (25-30°C)

• Extended expression periods (18 hours)

Purification Strategy:
Based on protocols established for His-tagged proteins from D. turgidum :

• Cell Disruption:

  • Mechanical methods (French press, sonication)

  • Chemical lysis (detergent-based methods like Cellytic B)

• Membrane Isolation:

  • Differential centrifugation to separate membrane fraction

  • Ultracentrifugation to collect membranes (100,000 × g)

• Solubilization:

  • Screen detergents systematically (DDM, LDAO, Fos-choline)

  • Test detergent concentration (critical micelle concentration × 2-5)

  • Include stabilizing additives (glycerol 10-20%)

• Chromatography Sequence:

  • IMAC (immobilized metal affinity chromatography) for His-tagged proteins

  • Size exclusion chromatography to separate monomers from aggregates

  • Ion exchange chromatography for further purification

• Quality Control Assessment:

  • SDS-PAGE for purity

  • Western blotting for identity confirmation

  • Dynamic light scattering for aggregation analysis

  • Circular dichroism for secondary structure verification

Reconstitution Methods:
For functional studies, reconstitution into lipid environments is often necessary:

• Detergent dialysis into liposomes

• Rapid dilution approach

• Direct incorporation into nanodiscs

When working with D. turgidum proteins, maintaining protein stability during purification is crucial - inclusion of osmolytes (glycerol, sucrose) and performing certain steps at elevated temperatures may help preserve native structure.

What bioinformatic approaches can predict functional properties of D. turgidum ATP synthase based on genomic data?

The complete genome sequence of D. turgidum (GenBank: CP001251.1) provides a foundation for comprehensive bioinformatic analysis of its ATP synthase components . Several computational approaches can yield valuable insights:

Comparative Genomic Analysis:

• Identify all ATP synthase genes in the D. turgidum genome

• Compare synteny and gene organization with other thermophiles

• Analyze conservation patterns across diverse thermophilic lineages

Phylogenetic Analysis:

• Construct phylogenetic trees for each ATP synthase subunit

• Identify lineage-specific adaptations

• Determine evolutionary relationships to other bacterial ATP synthases

Structural Prediction:

• Generate homology models based on known ATP synthase structures

• Validate models using energy minimization and Ramachandran analysis

• Identify unique structural features through comparative analysis

Molecular Dynamics Simulations:

• Simulate protein behavior at different temperatures

• Identify stabilizing interactions maintained at high temperatures

• Model conformational changes during catalytic cycle

Coevolution Analysis:

• Detect correlated mutations indicating functional constraints

• Identify potential interaction surfaces between subunits

• Predict critical residues for function and assembly

Relevant Databases and Tools:

The unusually low G+C content (33.96%) of D. turgidum despite its thermophilic lifestyle presents an interesting case for computational analysis of codon usage patterns and their potential impact on protein stability and expression.

How can researchers analyze the thermostability mechanisms of D. turgidum ATP synthase components?

Analyzing thermostability mechanisms requires integration of experimental data with computational approaches to identify key stabilizing features:

Experimental Methods for Thermostability Analysis:

Thermal Shift Assays:

• Differential scanning fluorimetry (DSF/Thermofluor)

• Differential scanning calorimetry (DSC)

• Circular dichroism (CD) with temperature ramping

Activity-Based Thermal Profiling:

• Measure enzyme activity after thermal challenge

• Determine temperature-activity relationships

• Calculate activation/inactivation energies

Structural Stability Analysis:

• Limited proteolysis at different temperatures

• Hydrogen-deuterium exchange mass spectrometry

• Intrinsic fluorescence monitoring

Computational Analysis Methods:

Sequence-Based Analysis:

• Amino acid composition comparison with mesophilic homologs

• Prediction of stabilizing interactions (salt bridges, H-bonds)

• Disorder prediction and flexibility analysis

Structural Analysis:

• Cavity and packing analysis

• Electrostatic interaction networks

• Hydrophobic core assessment

Molecular Dynamics:

• Protein behavior simulation at elevated temperatures

• Identification of regions with increased mobility

• Analysis of water interaction networks

Integrated Analysis Approach:

The presence of reverse gyrase in D. turgidum, typically associated with hyperthermophiles , suggests additional mechanisms may be involved in genome and protein stability that could inform the analysis of ATP synthase thermostability.

What are the main challenges in characterizing membrane proteins from extremophiles and how can they be addressed?

Working with membrane proteins from extremophiles like D. turgidum presents several unique challenges:

Challenge 1: Expression and Purification Difficulties

• Problem: Membrane proteins often express poorly and form inclusion bodies

• Solution Approaches:

  • Use specialized expression strains (C41/C43, Lemo21)

  • Express as fusion proteins with solubility-enhancing tags

  • Reduce expression rate through lower temperatures and inducer concentrations

  • Consider cell-free expression systems with added lipids/detergents

Challenge 2: Maintaining Native Conformation

• Problem: Detergent solubilization can disrupt native structure

• Solution Approaches:

  • Screen multiple detergent types and concentrations systematically

  • Use milder solubilization agents (SMA copolymer, amphipols)

  • Reconstitute into nanodiscs or liposomes for functional studies

  • Apply native mass spectrometry to verify intact complexes

Challenge 3: Thermostability Assessment

• Problem: Standard assays may not work at extremophile temperatures

• Solution Approaches:

  • Develop temperature-resistant assay components

  • Use thermostable coupling enzymes from other thermophiles

  • Employ physical methods (DSC, CD) rather than activity-based assays

  • Design specialized microfluidic or optical approaches

Challenge 4: Functional Reconstitution

• Problem: Regenerating function after purification

• Solution Approaches:

  • Use thermostable lipids for reconstitution

  • Optimize lipid composition based on D. turgidum membrane analysis

  • Apply droplet-based single-molecule approaches

  • Develop coupled enzyme systems that function at high temperatures

Methodological Protocol for Detergent Screening:

• Prepare small-scale membrane preparations

• Test panel of 8-10 detergents at multiple concentrations

• Assess protein extraction efficiency by Western blot

• Evaluate protein quality by size exclusion chromatography

• Validate function of best candidates with activity assays

These systematic approaches help overcome the significant challenges associated with working with thermophilic membrane proteins like D. turgidum ATP synthase components.

How can researchers differentiate between the ATP synthase components of D. turgidum and contaminants during purification?

Ensuring purity of recombinant ATP synthase components is critical for subsequent structural and functional studies. Several complementary approaches can be employed:

Chromatographic Approaches:

• IMAC Optimization: For His-tagged proteins, optimize imidazole gradient to separate contaminants

• Size Exclusion Chromatography: Separate based on size differences

• Ion Exchange Chromatography: Exploit charge differences between target and contaminants

Analytical Methods for Purity Assessment:

Functional Authentication Methods:

• Activity Assays: Verify expected biochemical activity

• Thermal Stability Testing: Compare to expected thermostability profile

• Proteomic Analysis: Peptide mass fingerprinting for identity confirmation

Contaminant Removal Strategies:

• Thermostability-Based Separation: Heat treatment to denature E. coli proteins

• Detergent-Specific Purification: Optimize detergent conditions for target protein

• Gradient Ultracentrifugation: For membrane protein-detergent complexes

Quality Control Criteria:

• Purity >95% by densitometry of SDS-PAGE

• Single peak by size exclusion chromatography

• Consistent activity characteristics across purification batches

• Mass spectrometry confirmation of identity

Utilizing protocols established for other D. turgidum proteins , a purification strategy using His-tag affinity chromatography followed by size exclusion chromatography under optimized detergent conditions provides an effective approach for obtaining pure ATP synthase components.

What are the potential applications of recombinant D. turgidum ATP synthase components in biotechnology and structural biology?

Recombinant ATP synthase components from extremophiles like D. turgidum offer unique properties with several promising applications:

Biotechnological Applications:

Biocatalysis and Enzyme Engineering:

• Template for designing thermostable ATP-producing systems

• Blueprint for engineering pH-resistant proton-pumping complexes

• Model for membrane protein stabilization techniques

Nanobiotechnology:

• Components for bio-nanomotors functioning at high temperatures

• Template for artificial proton-gradient devices

• Building blocks for synthetic metabolic systems

Structural Biology Research:

• Model system for studying membrane protein thermostability

• Platform for investigating energy coupling mechanisms

• Template for computational design of stable membrane proteins

Comparative Biochemistry:

• Understanding evolutionary adaptations to extreme environments

• Elucidating minimal requirements for ATP synthase function

• Investigating convergent evolution in thermophilic proteins

Future Research Directions:

Structure Determination:

• High-resolution cryo-EM structures of intact D. turgidum ATP synthase

• Comparative analysis with mesophilic homologs to identify stabilizing features

• Time-resolved structural studies of the catalytic cycle

Evolutionary Biology:

• Phylogenomic analysis of ATP synthase across the thermophilic spectrum

• Ancestral sequence reconstruction for evolutionary trajectories

• Horizontal gene transfer analysis in thermophiles

Bioengineering:

• Creation of hybrid ATP synthases with optimized properties

• Development of simplified, robust energy-transducing systems

• Design of temperature-resistant bioenergetic devices

Methodological Advancements:

• Optimized expression systems for thermophilic membrane proteins

• New assays for high-temperature enzyme characterization

• Advanced computational methods for predicting membrane protein stability

The unique phylogenetic position of D. turgidum in the Dictyoglomi phylum makes its ATP synthase particularly valuable for comparative studies seeking to understand the diversity of solutions that have evolved to maintain protein function at extreme temperatures.