Recombinant Dictyoglomus thermophilum ATP synthase subunit c (atpE)

What is the biological context of Dictyoglomus thermophilum ATP synthase subunit c (atpE)?

Dictyoglomus thermophilum ATP synthase subunit c (atpE) is a component of the F₀ sector of ATP synthase from the hyperthermophilic bacterium Dictyoglomus thermophilum. D. thermophilum belongs to the phylum Dictyoglomi, which comprises genetically distinct extremophiles that have been cultivated from anaerobic, hyperthermophilic hot spring environments, including the Tsuetate Hot Spring in Kumamoto Prefecture, Japan . The atpE protein functions as part of the membrane-embedded proton channel in ATP synthase, playing a critical role in the conversion of the proton gradient into mechanical energy that drives ATP synthesis. The protein consists of 84 amino acids and has several synonyms including ATP synthase F(0) sector subunit c, F-type ATPase subunit c, F-ATPase subunit c, and Lipid-binding protein .

What expression systems are most effective for producing recombinant D. thermophilum atpE?

Escherichia coli is the predominant expression system for recombinant D. thermophilum atpE production. As demonstrated in commercial preparations, the full-length protein (1-84 amino acids) can be successfully expressed with an N-terminal His-tag in E. coli . For optimal expression, researchers should consider the following methodological approaches:

• Vector selection: Vectors with strong, inducible promoters such as T7 or rhamnose promoters have proven effective for thermophilic proteins

• Codon optimization: Adapting the D. thermophilum sequence to E. coli codon usage can significantly improve expression

• Expression conditions: Growth at lower temperatures (15-25°C) after induction often improves proper folding

• Host strain: E. coli strains designed for expression of membrane proteins (e.g., C41/C43) may be beneficial since atpE is naturally membrane-associated

Similar approaches have been successful for other D. thermophilum proteins, such as DNA polymerase I, which was cloned into a rhamnose promoter vector with an N-terminal histidine tag and transformed into competent E. coli cells .

What are the optimal storage and reconstitution protocols for recombinant D. thermophilum atpE?

Based on established protocols for recombinant D. thermophilum atpE, researchers should follow these methodological guidelines for optimal storage and reconstitution:

Before opening, briefly centrifuge the vial to bring contents to the bottom. After reconstitution, the addition of glycerol and proper aliquoting are critical steps to maintain protein integrity over multiple uses .

How does the structure of D. thermophilum atpE contribute to its thermostability?

The amino acid sequence of D. thermophilum atpE (MLAWVIIVSIITAGLSVALVGMNATKAQGNAAASALESVARQPEAGDQINRMLLFALAFIETIMIFTLTVALILLFANPLLGKL) reveals several structural features that likely contribute to its thermostability :

• High proportion of hydrophobic residues: The sequence contains numerous valine, isoleucine, leucine, and alanine residues, which can form strong hydrophobic interactions stabilizing the protein core at high temperatures.

• Alpha-helical structure: The atpE subunit typically forms transmembrane alpha-helices in the membrane sector of ATP synthase. Alpha-helices are stabilized by hydrogen bonding patterns that can withstand thermal stress.

• Charged residue distribution: The sequence contains strategically positioned charged residues (glutamate, aspartate, arginine, lysine) that may form salt bridges, enhancing thermostability.

• Reduced flexibility: Thermophilic proteins often have reduced flexibility in regions not essential for function, limiting unfolding at high temperatures.

While D. thermophilum has an unusually low G+C content of 39.9% for a thermophile growing optimally at 72°C, it compensates with structural adaptations and the presence of specialized proteins like reverse gyrase, which is typically associated with hyperthermophiles .

How can researchers use D. thermophilum atpE as a model for studying ATP synthase in extremophiles?

Researchers can employ D. thermophilum atpE as a valuable model system for studying ATP synthase function in extremophiles through several methodological approaches:

• Comparative structural biology: Compare the structure of D. thermophilum atpE with mesophilic homologs to identify thermostabilizing features. This can be accomplished through crystallography, cryo-EM, or computational modeling.

• Site-directed mutagenesis: Using techniques similar to those employed for other proteins (like those described for Mycobacterium tuberculosis atpE), researchers can introduce specific mutations to probe structure-function relationships . For example:

  • Homologous recombination approaches

  • CRISPR-Cas9 genome editing

  • PCR-based site-directed mutagenesis

• Functional reconstitution: Reconstituting the ATP synthase complex in liposomes or nanodiscs to measure proton translocation and ATP synthesis at different temperatures.

• Evolutionary studies: Comparing atpE across the Dictyoglomi phylum and related thermophiles to understand evolutionary adaptations to extreme environments, building on genomic analyses of D. thermophilum and D. turgidum that revealed their highly syntenic nature .

What purification strategies yield the highest purity and activity for recombinant D. thermophilum atpE?

For optimal purification of His-tagged recombinant D. thermophilum atpE, researchers should implement a multi-step protocol:

• Immobilized Metal Affinity Chromatography (IMAC):

  • Use Ni-NTA or Co-NTA columns

  • Equilibrate with buffer containing 10-20 mM imidazole to reduce non-specific binding

  • Elute with an imidazole gradient (50-300 mM)

  • Similar His-tag purification approaches have been successfully used for other D. thermophilum proteins

• Size Exclusion Chromatography (SEC):

  • Further purify based on molecular size

  • Separate monomeric atpE from aggregates and contaminants

  • Analyze fractions by SDS-PAGE to confirm purity (target: >90% purity)

• Membrane protein-specific considerations:

  • Include appropriate detergents (DDM, LDAO, or C₁₂E₈) for solubilization

  • Maintain detergent concentration above CMC throughout purification

  • Consider using fluorinated or steroid-based detergents for improved stability

• Quality control:

  • Verify purity by SDS-PAGE (should exceed 90%)

  • Confirm identity by mass spectrometry or Western blotting

  • Assess functional activity through ATP synthase reconstitution assays

What PCR-based methods can be used to clone and manipulate D. thermophilum atpE for research?

Researchers can employ several PCR-based techniques to effectively clone and manipulate D. thermophilum atpE, drawing on successful approaches used for other genes from this organism:

• Two-step PCR protocol: This approach has been successfully used for cloning genes from D. thermophilum, as demonstrated with xylanase genes . For atpE:

  • First amplify using consensus primers based on conserved regions

  • Then perform genomic walking PCR to obtain full-length sequence

• High-fidelity amplification:

  • Use thermostable DNA polymerases with proofreading activity (like Phusion)

  • Design primers that span the start and stop codons of atpE

  • Include appropriate restriction sites for subsequent cloning

• Expression vector construction:

  • Insert the amplified atpE into vectors with inducible promoters (e.g., rhamnose promoter)

  • Include affinity tags (His-tag) for purification

  • Transform into appropriate E. coli host strains

• PCR-based site-directed mutagenesis:

  • Design overlapping primers containing desired mutations

  • Perform PCR amplification to generate mutant constructs

  • Confirm mutations by Sanger sequencing

• Recombineering approaches:

  • For more complex genetic manipulations, techniques like homologous recombineering with ssDNA oligonucleotides can be adapted from methods used for other bacterial species

How can researchers assess the thermostability and activity of purified D. thermophilum atpE?

To comprehensively evaluate the thermostability and activity of purified D. thermophilum atpE, researchers should implement the following methodological approaches:

• Thermal shift assays:

  • Differential scanning fluorimetry (DSF) with SYPRO Orange dye

  • Differential scanning calorimetry (DSC) to determine melting temperature (Tm)

  • Circular dichroism (CD) spectroscopy at increasing temperatures to monitor secondary structure changes

• Functional reconstitution assays:

  • Reconstitute atpE into liposomes with other ATP synthase components

  • Measure proton translocation using pH-sensitive fluorescent dyes

  • Assess ATP synthesis activity at different temperatures (25-85°C)

• Protein stability assessments:

  • Incubate protein at various temperatures (60-90°C) for different time periods

  • Analyze remaining structural integrity by SDS-PAGE or native-PAGE

  • Determine half-life at different temperatures

• Comparative activity assays:

  • Compare with atpE proteins from mesophilic organisms

  • Assess relative activity across a temperature gradient

  • Determine temperature optima and activation energy

• Structural analysis:

  • Monitor structural integrity at different temperatures using spectroscopic methods

  • Assess oligomeric state stability using size exclusion chromatography or analytical ultracentrifugation

These approaches can provide comprehensive data on the thermostability profile and structure-function relationships of D. thermophilum atpE, similar to analyses performed for other thermostable enzymes from Dictyoglomus species .

What methodological challenges must researchers overcome when working with recombinant D. thermophilum atpE?

Researchers face several significant methodological challenges when working with D. thermophilum atpE:

What are the critical differences in experimental approaches for studying atpE in thermophiles versus mesophiles?

When studying atpE in thermophiles like D. thermophilum compared to mesophiles, researchers must adapt their experimental approaches in several critical ways:

• Temperature considerations:

  • Enzymatic assays must be conducted at elevated temperatures (70-85°C) to observe optimal activity

  • Control experiments should include activity profiles across broad temperature ranges

  • Specialized water baths, thermocyclers, or incubators capable of stable high temperatures are required

• Buffer and reagent stability:

  • Standard biochemical buffers may degrade at high temperatures

  • Thermostable alternatives to common reagents must be identified

  • pH changes with temperature must be accounted for (ΔpKa/ΔT effect)

• Protein purification modifications:

  • Heat treatment steps (60-70°C) can be included to remove mesophilic contaminants

  • Chromatography steps may need to be performed at elevated temperatures

  • More stringent precautions against protease degradation may be necessary

• Structural stability assessment:

  • Reference temperatures for stability studies must be adjusted upward

  • Thermal denaturation studies need extended temperature ranges

  • Comparative studies should include proteins from organisms with different thermal optima

• Functional reconstitution adaptations:

  • Lipid composition may need to be adjusted to maintain membrane fluidity at high temperatures

  • More thermostable fluorescent probes for functional assays

  • Modified reaction kinetics accounting for increased thermal energy

These methodological differences are essential for accurate characterization of thermophilic proteins, as demonstrated in studies of other D. thermophilum enzymes like xylanases, which exhibited optimal activity at 85°C .

How might structural studies of D. thermophilum atpE inform drug development targeting bacterial ATP synthases?

Structural studies of D. thermophilum atpE have significant potential to inform novel antimicrobial development through several research avenues:

• Comparative structural biology approach:

  • Detailed structural characterization of D. thermophilum atpE could reveal conserved binding pockets present across bacterial species

  • Comparing thermophilic and pathogenic bacterial ATP synthases may identify targetable differences from human ATP synthases

  • Studies of extremophilic proteins often reveal rigid structural elements that can serve as stable drug-binding sites

• Drug resistance mechanism elucidation:

  • Research on mutations in mycobacterial atpE (like Ile66Val) has shown their role in bedaquiline resistance

  • Similar mutation studies in D. thermophilum atpE could provide insights into resistance mechanisms

  • Computational modeling methods that demonstrated how atpE Ile66Val mutation minimally disrupts bedaquiline-ATP synthase interaction could be applied to other potential inhibitors

• Thermostability-focused drug design:

  • Understanding how D. thermophilum atpE maintains functionality at high temperatures could inform the design of thermostable drugs

  • Identify binding sites that remain accessible despite thermostabilizing adaptations

  • Potential development of inhibitors that exploit unique structural features of bacterial ATP synthases

• Bayesian approach applications:

  • Methods used to estimate the probability of bedaquiline resistance for different atpE mutations could be applied to predict cross-resistance patterns

  • Development of statistical models to predict mutation effects on drug binding

What emerging technologies could advance our understanding of D. thermophilum atpE function and assembly?

Several cutting-edge technologies hold promise for addressing current knowledge gaps regarding D. thermophilum atpE:

• Cryo-electron microscopy advances:

  • Single-particle cryo-EM could resolve high-resolution structures of the complete ATP synthase complex

  • Cryo-electron tomography might visualize ATP synthase in native-like membrane environments

  • Time-resolved cryo-EM could potentially capture different conformational states during the catalytic cycle

• Advanced molecular dynamics simulations:

  • Enhanced sampling techniques could model conformational changes at high temperatures

  • Specialized force fields for thermophilic proteins might improve simulation accuracy

  • Coarse-grained simulations could extend timescales to observe complete rotary cycles

• Native mass spectrometry:

  • Could determine subunit stoichiometry and assembly intermediates

  • May detect post-translational modifications and lipid interactions

  • Would provide insights into the stability of subcomplexes at different temperatures

• Single-molecule techniques:

  • FRET studies could track conformational changes during proton translocation

  • Magnetic tweezers or optical traps might measure rotary motion at different temperatures

  • Single-molecule force spectroscopy could assess stability of individual subunits

• Next-generation genome editing:

  • CRISPR-Cas systems optimized for extremophiles could enable direct genetic manipulation

  • Precise mutagenesis approaches similar to the homologous recombineering methods used in other bacterial species

  • Development of thermostable selection markers for genetic studies

These technologies would complement existing approaches and potentially overcome current methodological limitations in studying thermophilic membrane protein complexes.

How might comparative genomics inform our understanding of ATP synthase evolution in extremophiles?

Comparative genomic approaches offer powerful tools for understanding the evolutionary adaptations of ATP synthase in extremophiles like D. thermophilum:

• Phylogenomic analysis:

  • The Dictyoglomi phylum, comprising D. thermophilum and D. turgidum, is genetically distinct and divergent from known taxa

  • Genomic analyses have shown that the two Dictyoglomus genomes are highly syntenic and distantly related to Caldicellulosiruptor species

  • Expanded phylogenetic studies including newly discovered extremophiles could reveal convergent evolutionary pathways

• Horizontal gene transfer investigation:

  • Analysis of atpE sequence conservation and genomic context across diverse thermophiles

  • Identification of potential gene transfer events that contributed to thermophilic adaptations

  • Assessment of atpE coevolution with other ATP synthase components

• Evolutionary rate analysis:

  • Despite optimal growth at 72°C, D. thermophilum has an anomalously low G+C content of 39.9%

  • Comparative studies could reveal whether atpE follows the same unusual evolutionary pattern

  • Identification of positively selected sites that confer thermostability

• Structural genomics approach:

  • Comparison of predicted secondary and tertiary structures across phylogenetic lineages

  • Identification of conserved structural elements despite sequence divergence

  • Analysis of compensatory mutations that maintain protein stability

• Ecological genomics integration:

  • Correlation of genomic features with environmental parameters of source habitats

  • D. thermophilum was isolated from hot springs in Japan , and comparative studies with organisms from different thermal environments could reveal habitat-specific adaptations

  • Investigation of how environmental pressures shape ATP synthase evolution

Such comparative genomic approaches could provide fundamental insights into the molecular adaptations that enable ATP synthase to function in extreme environments, with potential applications in protein engineering and synthetic biology.