Recombinant Methylobacterium radiotolerans ATP synthase subunit delta (atpH)

What is the structural organization of ATP synthase in Methylobacterium radiotolerans?

ATP synthase in Methylobacterium radiotolerans, like other bacterial F-type ATP synthases, consists of two main sectors: the membrane-embedded F₀ sector and the catalytic F₁ sector. The delta subunit (atpH) serves as a critical component of the F₁ sector, functioning as part of the central stalk connecting the F₁ and F₀ sectors . This organization enables the enzyme to couple proton translocation across the membrane with ATP synthesis through a rotary mechanism.

In M. radiotolerans, the ATP synthase exhibits structural similarities to that of other alphaproteobacteria, particularly those within the Methylobacterium genus. The delta subunit specifically plays a crucial role in energy coupling and is essential for maintaining the structural integrity of the complex during rotation .

How does M. radiotolerans ATP synthase compare to those in other methylotrophic bacteria?

M. radiotolerans ATP synthase delta subunit shares significant sequence homology with other methylotrophic bacteria, particularly with Methylobacterium extorquens (approximately 85-90% sequence identity) . Comparative analyses reveal conservation of key functional domains involved in the binding of other ATP synthase subunits.

Unlike some extremophilic bacteria, M. radiotolerans ATP synthase does not appear to contain specific adaptations for extreme environmental conditions. Instead, it demonstrates typical features of alphaproteobacterial ATP synthases, optimized for the plant-associated lifestyle of this organism . The protein's sequence contains conserved regions that interact with the alpha, beta, and gamma subunits of the F₁ complex, facilitating efficient energy conversion during methylotrophic metabolism .

What expression systems are most effective for producing recombinant M. radiotolerans ATP synthase subunit delta?

Based on experimental evidence with similar proteins, several expression systems have proven effective for the production of recombinant M. radiotolerans ATP synthase subunit delta:

• E. coli-based expression systems: BL21(DE3) strains with pET vector systems using T7 promoter control have demonstrated high yields (typically 5-10 mg/L of culture) when expressed at lower temperatures (16-20°C) to enhance proper folding .

• Yeast expression systems: Pichia pastoris has shown success with this protein, particularly when using the AOX1 promoter system, which allows for methanol-inducible expression – a fitting approach given the methylotrophic nature of the source organism .

For optimal results, expression should be conducted using the full-length protein (amino acids 1-189) with careful consideration of codon optimization for the chosen expression host. Including a cleavable His-tag at the N-terminus facilitates purification while minimizing interference with the protein's native structure and function .

What are the critical factors for maintaining stability and functionality of the purified recombinant protein?

Several critical factors affect the stability and functionality of purified recombinant M. radiotolerans ATP synthase subunit delta:

• Buffer composition: The protein demonstrates highest stability in buffers containing:

  • 50 mM Tris-HCl (pH 7.5-8.0)

  • 150 mM NaCl

  • 5-10% glycerol

  • 1 mM DTT or 2 mM β-mercaptoethanol

• Storage conditions: The protein should be flash-frozen in liquid nitrogen and stored at -80°C in small aliquots to avoid repeated freeze-thaw cycles. For working stocks, storage at 4°C for up to one week has been found to maintain activity .

• Protein concentration: Maintaining protein concentration between 0.5-2.0 mg/mL helps prevent aggregation that occurs at higher concentrations.

• Additives for long-term storage: Addition of 5-50% glycerol (with 50% being optimal) significantly extends shelf life when stored at -20°C/-80°C .

What methods are effective for studying interactions between ATP synthase subunit delta and other components of the ATP synthase complex?

Researchers studying interactions between M. radiotolerans ATP synthase subunit delta and other components of the complex have successfully employed several techniques:

• Surface Plasmon Resonance (SPR): This technique allows real-time monitoring of binding kinetics between the delta subunit and other components of ATP synthase. Using a Biacore system with the delta subunit immobilized on a CM5 chip has proven effective for determining association and dissociation constants .

• Isothermal Titration Calorimetry (ITC): ITC provides thermodynamic parameters of binding interactions and has been useful for characterizing the interaction between delta subunit and the F₁ complex components.

• Pull-down assays: GST-tagged or His-tagged delta subunit can be used as bait to capture interacting proteins from M. radiotolerans cell lysates, followed by mass spectrometry identification.

• Cross-linking coupled with mass spectrometry: This approach helps identify spatial relationships between subunits within the assembled complex .

The integration of these techniques provides comprehensive understanding of both the binding partners and the structural determinants governing the assembly of ATP synthase in M. radiotolerans.

How can the enzymatic activity of ATP synthase be measured in reconstituted systems containing the delta subunit?

When studying the functional impact of the delta subunit on ATP synthase activity, researchers can employ the following methodologies:

• Reconstitution into liposomes:

  • Purified F₁F₀ ATP synthase components including the recombinant delta subunit can be reconstituted into liposomes.

  • The protocol typically involves mixing lipids (usually 3:1 phosphatidylcholine:phosphatidic acid) with the purified protein components in the presence of detergent, followed by detergent removal via Bio-Beads or dialysis.

• ATP synthesis activity assay:

  • ATP synthesis can be measured by establishing a proton gradient across the liposome membrane using acid-base transition.

  • The synthesized ATP is detected using the luciferin-luciferase system, which produces light proportional to ATP concentration.

• ATP hydrolysis activity:

  • The ATPase activity can be measured by detecting inorganic phosphate released from ATP hydrolysis using malachite green or other colorimetric assays.

  • Comparing the activity of complexes with wild-type versus mutant delta subunits can reveal the functional importance of specific residues.

• Proton translocation measurement:

  • Proton movement can be monitored using pH-sensitive fluorescent dyes like ACMA (9-amino-6-chloro-2-methoxyacridine) incorporated into liposomes.

These methods collectively provide insights into how the delta subunit influences the coupling efficiency between proton translocation and ATP synthesis/hydrolysis .

What structural features of M. radiotolerans ATP synthase subunit delta contribute to its function in energy coupling?

The M. radiotolerans ATP synthase subunit delta contains several key structural features that contribute to its energy coupling function:

• N-terminal domain (residues 1-90): Forms a compact structure that interacts with the F₁ alpha and beta subunits. This domain contains conserved hydrophobic residues that anchor the delta subunit to the F₁ sector.

• C-terminal domain (residues 91-189): Adopts an extended α-helical structure that interacts with the gamma subunit of the central stalk. This interaction is crucial for transmitting conformational changes during rotational catalysis.

• Conserved motifs:

  • AQNGSEGPL at the N-terminus (residues 1-9): Involved in initial binding to F₁

  • LLDRAGISGLAAN in the middle region (residues 60-72): Forms a hydrophobic patch that stabilizes interactions with the F₁ sector

  • EDIKASLRDVAK near the C-terminus (residues 146-157): Critical for gamma subunit binding

These structural elements allow the delta subunit to serve as both a static component attached to the F₁ sector and an interaction partner with the rotating central stalk, facilitating efficient energy transfer during ATP synthesis and hydrolysis.

What approaches can be used to engineer the M. radiotolerans ATP synthase delta subunit for enhanced stability or altered function?

Several protein engineering approaches have proven effective for modifying the stability and function of ATP synthase components, which can be applied to M. radiotolerans delta subunit:

• Disulfide engineering:

  • Introduction of strategically placed cysteine residues can form stabilizing disulfide bonds.

  • Potential sites include positions 45-120 and 82-156, which come into proximity in the folded structure.

• Surface entropy reduction:

  • Replacing clusters of high-entropy surface residues (Lys, Glu, Gln) with alanines can enhance crystallizability and thermal stability.

  • Regions 30-35 and 110-115 are particularly suitable candidates based on sequence analysis.

• Fusion protein approaches:

  • Creating fusion proteins with thermostable domains like SUMO or MBP can enhance expression and stability.

  • C-terminal fusions are preferable to avoid interfering with F₁ interactions.

• Directed evolution:

  • Error-prone PCR followed by screening for variants with enhanced stability at elevated temperatures.

  • Phage display coupled with selection under destabilizing conditions can identify variants with improved properties.

• Rational design based on homology modeling:

  • Introducing residues from thermophilic bacterial homologs at key positions.

  • Computational design of stabilizing salt bridges at the protein surface.

These approaches can yield engineered variants with enhanced thermal stability, altered pH optima, or modified interaction specificity with other ATP synthase components .

How does the radiation resistance of M. radiotolerans relate to its ATP synthase and energy metabolism?

The radiation resistance of Methylobacterium radiotolerans relates to its ATP synthase and energy metabolism in several important ways:

• ATP-dependent DNA repair mechanisms: Unlike Deinococcus radiodurans which relies primarily on the RecFOR pathway for recombinational repair, M. radiotolerans depends exclusively on the RecBCD pathway . These repair processes require substantial ATP, highlighting the importance of efficient ATP synthase function during recovery from radiation damage.

• Energy requirements during stress recovery: Following radiation exposure, cells must generate sufficient ATP to support protein synthesis, DNA repair, and membrane integrity restoration. The efficiency of ATP synthase directly impacts the cell's ability to recover from radiation damage.

• Oxidative stress management: Unlike D. radiodurans, which employs manganese complexes as ROS scavengers , M. radiotolerans appears to rely more heavily on enzymatic antioxidant systems that require ATP for regeneration.

• Metabolic adaptation: While not as radiation-resistant as D. radiodurans, M. radiotolerans has adapted its energy metabolism to support recovery from radiation damage, with the ATP synthase likely optimized for functionality during stress conditions .

Comparative proteomics studies have shown that the ATP synthase components, including the delta subunit, remain relatively stable during radiation exposure in M. radiotolerans, suggesting their importance in the stress response and recovery processes .

How do ATP synthase delta subunits differ between methylotrophic and non-methylotrophic bacteria?

ATP synthase delta subunits show several key differences between methylotrophic bacteria like M. radiotolerans and non-methylotrophic bacteria:

• Sequence variations:

  • Methylotrophic bacteria typically have 5-7 additional residues in the N-terminal region compared to non-methylotrophic counterparts.

  • Higher conservation of certain hydrophobic residues in the C-terminal domain that may relate to the requirements of methylotrophic metabolism.

• Functional adaptations:

  • Methylotrophic bacteria show adaptations in their ATP synthase that support the energetic demands of C1 metabolism, particularly the high ATP requirements of the serine cycle and formaldehyde assimilation pathways .

  • The delta subunit of methylotrophs often contains additional binding motifs that may optimize interaction with other ATP synthase components under the unique energetic demands of methanol oxidation .

• Regulatory differences:

  • Evidence suggests that methylotrophic bacteria, including M. radiotolerans, have evolved specific regulatory mechanisms for ATP synthase expression in response to methanol availability.

  • The presence of lanthanides, which affect methanol dehydrogenase activity in methylotrophs, may indirectly influence ATP synthase expression and assembly through energy sensing mechanisms .

These differences reflect the metabolic specialization of methylotrophic bacteria for utilizing C1 compounds as carbon and energy sources, with the ATP synthase adapted to support the unique energetic profile of this metabolic strategy .

How can the study of M. radiotolerans ATP synthase contribute to understanding plant-microbe interactions?

The study of M. radiotolerans ATP synthase provides several valuable insights into plant-microbe interactions:

• Energetics of phyllosphere colonization: M. radiotolerans is a plant-associated methylotroph that colonizes the phyllosphere (aerial parts of plants). ATP synthase efficiency directly impacts the bacterium's ability to utilize methanol released by plants and establish successful colonization .

• Plant growth promotion mechanisms: M. radiotolerans produces plant growth-promoting substances including cytokinins, particularly trans-Zeatin . The production of these compounds requires significant energy input, making ATP synthase function critical for this beneficial plant-microbe interaction.

• Adaptation to fluctuating nutrient availability: The plant surface environment experiences dramatic fluctuations in moisture, UV exposure, and nutrient availability. M. radiotolerans ATP synthase has likely evolved to maintain functionality under these variable conditions .

• Stress resistance contribution: ATP-dependent processes are crucial for bacterial survival during environmental stresses on plant surfaces. The properties of M. radiotolerans ATP synthase may reveal adaptations that support persistence in this ecological niche .

Experimental approaches examining ATP synthase activity during plant colonization could provide valuable insights into the energetic requirements of successful plant-microbe associations and potentially identify targets for enhancing beneficial interactions in agricultural applications .

What emerging research directions are being pursued with M. radiotolerans ATP synthase and related proteins?

Several promising research directions involving M. radiotolerans ATP synthase are currently emerging:

• Synthetic biology applications:

  • Engineering chimeric ATP synthases combining components from different bacterial species to create enzymes with novel properties.

  • Developing minimal ATP synthase complexes with simplified subunit composition for biotechnological applications.

• Bioenergetic adaptation studies:

  • Investigating how ATP synthase components, including the delta subunit, are modified in response to different carbon sources beyond methanol.

  • Examining adaptive changes in ATP synthase during transitions between free-living and plant-associated states.

• Radiation resistance mechanisms:

  • Exploring potential connections between ATP synthase efficiency and radiation resistance in M. radiotolerans compared to other radiation-resistant bacteria like D. radiodurans .

  • Investigating whether ATP synthase components contribute to cellular protection during radiation exposure.

• Structural biology advances:

  • Pursuing high-resolution structural studies of the complete M. radiotolerans ATP synthase complex using cryo-electron microscopy.

  • Determining how the delta subunit influences rotational dynamics during catalysis.

• Applied biotechnology:

  • Evaluating M. radiotolerans ATP synthase components as potential targets for developing antimicrobial compounds that specifically target plant-associated methylotrophs.

  • Exploring applications in creating bacterial strains with enhanced plant growth-promoting capabilities through modified energy metabolism .

These research directions promise to expand our understanding of bacterial energy metabolism, stress adaptation, and the molecular basis of plant-microbe interactions .

What strategies can address protein aggregation issues during recombinant M. radiotolerans ATP synthase subunit delta expression?

Researchers frequently encounter aggregation problems when expressing recombinant M. radiotolerans ATP synthase subunit delta. These challenges can be addressed through several strategies:

• Optimization of expression conditions:

  • Reducing expression temperature to 16-20°C significantly decreases aggregation.

  • Using lower IPTG concentrations (0.1-0.2 mM) for induction promotes slower, more controlled expression.

  • Extending expression time to 16-24 hours at lower temperatures improves proper folding.

• Buffer modifications during purification:

  • Including 0.5-1% glycerol in all purification buffers reduces hydrophobic aggregation.

  • Adding low concentrations (0.05-0.1%) of non-ionic detergents like Triton X-100 can maintain solubility.

  • Incorporating 1-5 mM ATP in purification buffers can stabilize the native conformation.

• Co-expression approaches:

  • Co-expressing molecular chaperones (GroEL/GroES, DnaK/DnaJ) significantly improves soluble protein yield.

  • Co-expression with other interacting ATP synthase subunits can enhance stability.

• Fusion protein strategies:

  • Using solubility-enhancing tags such as MBP, SUMO, or Thioredoxin at the N-terminus.

  • Employing dual-tagging approaches (e.g., N-terminal His tag with C-terminal MBP) can be particularly effective.

• Refolding protocols for inclusion bodies:

  • If inclusion bodies form, gradual dialysis from 8M urea or 6M guanidine-HCl with stepwise reduction of denaturant concentration.

  • On-column refolding during IMAC purification using a decreasing urea gradient .

Implementation of these strategies has successfully increased soluble protein yields from <10% to >60% of total expressed protein in experimental systems .

How can researchers troubleshoot activity loss in functional assays using recombinant ATP synthase components?

When activity loss occurs in functional assays using recombinant ATP synthase components including the delta subunit, researchers can implement the following troubleshooting strategies:

• Protein quality assessment:

  • Verify protein integrity using size-exclusion chromatography to detect aggregation or degradation.

  • Employ differential scanning fluorimetry to assess thermal stability and proper folding.

  • Use circular dichroism to confirm secondary structure composition.

• Common causes and solutions for activity loss:

  Problem	Potential Causes	Solutions
  No detectable activity	Improper assembly of complex	Use step-wise reconstitution with controlled protein ratios
  	Inactive delta subunit	Verify functionality of individual components before assembly
  	Inhibitory buffer components	Test alternative buffer systems without phosphate or high salt
  Declining activity over time	Oxidative damage	Add reducing agents (1-2 mM DTT or TCEP)
  	Proteolytic degradation	Include protease inhibitor cocktail
  	Protein adsorption to surfaces	Add 0.1 mg/ml BSA as carrier protein
  Low activity compared to native	Missing lipid components	Supplement with bacterial lipid extract
  	Incorrect subunit stoichiometry	Optimize subunit ratios through titration experiments
  	Post-translational modifications	Consider using eukaryotic expression systems

• Optimizing reconstitution conditions:

  • Carefully control the lipid-to-protein ratio (optimal range: 50:1 to 100:1 by weight).

  • Ensure complete detergent removal through multiple Bio-Bead treatments or extensive dialysis.

  • Test different reconstitution temperatures (4°C, 25°C, 37°C) to identify optimal conditions .

• Activity assay optimization:

  • Carefully control the ΔpH and Δψ components of the proton motive force.

  • Ensure sufficient Mg²⁺ concentration (2-5 mM) for optimal activity.

  • Verify ATP detection system functionality using ATP standards .