Recombinant Enterococcus faecalis ATP synthase subunit delta (atpH)

What expression systems are most effective for recombinant E. faecalis atpH production?

Efficient expression of recombinant E. faecalis atpH requires careful selection of expression systems based on research objectives:

• E. coli BL21(DE3) with pET vectors: Provides highest yield (3-5 mg/L) with N-terminal His6-tag for purification

• Expression conditions optimization:

  • Induction at OD600 0.6-0.8 with 0.5 mM IPTG

  • Post-induction growth at 30°C rather than 37°C improves solubility

  • Supplementation with 0.2% glucose reduces basal expression

  • Addition of 10% glycerol to lysis buffer enhances stability

For functional studies requiring native conformation, consider Gram-positive expression hosts such as Lactococcus lactis, which provides lower yield but potentially better folding of the target protein. When using E. coli systems, co-expression with molecular chaperones (GroEL/GroES) significantly improves solubility and reduces inclusion body formation.

What purification strategies yield highest purity recombinant atpH protein?

Purification of recombinant E. faecalis atpH requires a multi-step approach to achieve high purity and maintain functional integrity:

• Initial capture: IMAC (Immobilized Metal Affinity Chromatography) using Ni-NTA resin

  • Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT

  • Wash with 20-30 mM imidazole to remove non-specific binding

  • Elute with 250-300 mM imidazole gradient

• Intermediate purification: Ion-exchange chromatography

  • Dialyze against 20 mM Tris-HCl pH 7.5, 50 mM NaCl

  • Apply to anion exchange column (Q-Sepharose)

  • Elute with NaCl gradient (50-500 mM)

• Polishing step: Size exclusion chromatography

  • Superdex 75 column in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

  • Collect monomeric fractions (typically eluting at ~45-50 ml on standard 120 ml columns)

Critical factors affecting purification success include maintaining reducing conditions throughout the process, avoiding extreme pH conditions, and using glycerol as a stabilizing agent. Tag removal using TEV protease may be necessary for certain functional or structural studies.

How can researchers assess the functional integrity of purified recombinant atpH?

Evaluating the functional integrity of recombinant E. faecalis atpH requires multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to verify secondary structure content

  • Thermal shift assays to determine stability and proper folding

  • Limited proteolysis to confirm compact domain structure

• Binding assays with partner subunits:

  • Microscale thermophoresis to measure binding affinities with α, β, and γ subunits

  • Size exclusion chromatography with multi-angle light scattering to verify complex formation

  • Pull-down assays to confirm interaction with other ATP synthase components

• Functional reconstitution:

  • Integration into liposomes with other ATP synthase components

  • ATP synthesis measurements using luciferin-luciferase assays under ion gradients

  • Proton/sodium translocation assays using pH-sensitive or Na+-sensitive fluorescent probes

Importantly, the delta subunit functions as part of the central stalk connecting F1 and F0 domains, so its activity cannot be measured in isolation but must be assessed in the context of the assembled complex or through interactions with partner subunits.

How does E. faecalis atpH differ structurally and functionally from sodium-driven ATP synthases like those in E. hirae?

The structural and functional distinctions between E. faecalis atpH (H+-driven) and E. hirae ATP synthase (Na+-driven) reflect fundamental adaptations to different bioenergetic strategies:

• Structural differences:

  • E. faecalis atpH lacks specific Na+-binding motifs present in E. hirae

  • Comparative structural analysis shows differences in the C-terminal domain that interacts with the γ subunit

  • E. hirae V-ATPase contains specialized residues that coordinate Na+ during catalysis

• Ion specificity determinants:

  • Key residues in the coupling mechanism differ: E. faecalis utilizes protonatable residues (Asp, Glu) at critical positions

  • E. hirae contains specific Na+-binding sites with coordination geometry optimized for sodium

  • These differences manifest in ion-dependence of ATP synthesis activity: E. hirae shows optimal activity at high Na+ concentrations while E. faecalis responds to proton gradients

• Kinetic parameters comparison:

  • E. hirae V-ATPase synthesizes ATP at 4.7 s-1 under high sodium motive force (269.3 mV)

  • E. faecalis F-type ATP synthase typically exhibits different kinetic parameters, reflecting its adaptation to proton-driven synthesis

  • At equilibrium, both ion gradients (ΔpNa or ΔpH) and membrane potential (Δψ) contribute to ATP synthesis

These differences highlight evolutionary adaptations to different ecological niches and energy sources, with important implications for bacterial physiology and potential antimicrobial targets.

What role does atpH play in E. faecalis stress response and adaptation to changing environments?

The ATP synthase delta subunit (atpH) contributes significantly to E. faecalis stress response through several mechanisms:

• Acid stress adaptation:

  • Under acidic conditions, E. faecalis modulates ATP synthase activity to maintain intracellular pH

  • The delta subunit undergoes conformational changes that affect coupling efficiency

  • E. faecalis shifts between oxidative phosphorylation and fermentation depending on external pH

  • Lactic acid production through LDH becomes a significant ATP source under acidic conditions

• Nutrient limitation response:

  • During carbon source limitation, E. faecalis can utilize alternative pathways for ATP generation

  • The agmatine deiminase pathway generates ATP through substrate-level phosphorylation

  • atpH regulation helps balance energy production between F-type ATP synthase and substrate-level phosphorylation

• Oxidative stress management:

  • ATP synthase activity influences membrane potential, affecting susceptibility to oxidative damage

  • atpH modifications can alter proton translocation efficiency, affecting ROS production

  • ATP availability determines cellular capacity to repair oxidative damage

• Biofilm formation and persistence:

  • ATP synthesis is critical for initial attachment and biofilm matrix production

  • E. faecalis biofilms show altered expression of ATP synthase components including atpH

  • Energy conservation through regulated ATP synthase activity supports long-term persistence

These adaptive responses highlight atpH's role beyond structural contribution to ATP synthase, positioning it as a key component in E. faecalis stress response networks.

How does atpH contribute to ATP synthesis regulation in E. faecalis, and what methods best characterize these regulatory mechanisms?

The delta subunit (atpH) plays crucial regulatory roles in E. faecalis ATP synthesis that can be characterized through sophisticated methodological approaches:

• Conformational regulation investigation:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify conformational changes under varying conditions

  • FRET-based assays using labeled atpH to detect real-time structural rearrangements

  • Single-molecule techniques to observe rotational dynamics during catalysis

  • Cryo-EM studies of ATP synthase in different catalytic states

• Protein-protein interactions characterization:

  • Crosslinking coupled with mass spectrometry to map interaction interfaces

  • Surface plasmon resonance to determine binding kinetics with other subunits

  • Co-immunoprecipitation experiments from native membranes to identify regulatory partners

  • Yeast two-hybrid or bacterial two-hybrid screens to discover novel interactors

• Post-translational modifications analysis:

  • Mass spectrometry to identify phosphorylation, acetylation, or other modifications

  • Site-directed mutagenesis of modified residues to assess functional impact

  • Antibodies against specific modifications to track regulatory changes

  • In vitro modification assays to determine effects on activity

• Integrated structural-functional approach:

  • Mutagenesis of key residues at subunit interfaces

  • Reconstitution experiments with modified components

  • ATP synthesis/hydrolysis measurements under varying conditions

  • Computational modeling of energy transfer through the complex

These methods reveal that atpH functions as a mechanical coupling element and a regulatory node that responds to cellular energetic status, adjusting ATP synthase activity to optimize energy conservation under different environmental conditions.

What site-directed mutagenesis strategies are most informative for studying E. faecalis atpH function?

Effective site-directed mutagenesis studies of E. faecalis atpH require strategic targeting of residues based on structure-function hypotheses:

• Targeting critical residues:

  • Interface residues between atpH and other subunits (particularly γ and α/β)

  • Conserved charged residues that may participate in energy transduction

  • Residues with predicted conformational flexibility acting as molecular hinges

  • Sites with potential post-translational modifications

• Mutation design principles:

  • Conservative substitutions (e.g., Asp→Glu) to test side chain length effects

  • Alanine scanning to remove side chain contributions entirely

  • Charge reversals (e.g., Lys→Glu) to probe electrostatic interactions

  • Cysteine substitutions for crosslinking or labeling experiments

• Experimental validation workflow:

  • Expression level and solubility assessment

  • Thermal stability measurement using differential scanning fluorimetry

  • Binding affinity determination with partner subunits

  • Functional reconstitution and activity assays

• Data integration and interpretation:

  • Correlation of structural changes with functional effects

  • Comparison with homologous mutations in related species

  • Integration with computational models of ATP synthase dynamics

  • Classification of mutations as affecting assembly, catalysis, or regulation

This systematic approach has revealed key residues in the N-terminal domain that mediate critical interactions with the F1 head, and C-terminal residues that coordinate with the rotating γ subunit during catalysis.

What advanced structural biology techniques provide the most valuable insights into E. faecalis atpH structure and function?

Multiple complementary structural approaches reveal different aspects of E. faecalis atpH biology:

The most comprehensive understanding comes from integrating these approaches. For example, crystallography provides the static structure, HDX-MS reveals dynamic regions, and single-molecule techniques capture functional movements during ATP synthesis. Recent advances in time-resolved cryo-EM have been particularly valuable for capturing different conformational states of ATP synthase components during the catalytic cycle.

How can recombinant E. faecalis atpH be utilized to develop novel antimicrobial strategies?

Recombinant E. faecalis atpH offers multiple avenues for antimicrobial development:

• Target-based drug discovery approaches:

  • High-throughput screening of compound libraries against purified atpH

  • Fragment-based drug discovery to identify chemical starting points

  • Structure-based virtual screening using solved atpH structures

  • Rational design of peptide inhibitors targeting critical interfaces

• Functional assay development:

  • FRET-based binding assays for compounds disrupting atpH-γ subunit interactions

  • ATP synthesis inhibition assays using reconstituted systems

  • Whole-cell ATP depletion assays with compound treatment

  • Membrane potential disruption measurements in intact cells

• Selectivity determination methods:

  • Comparative binding studies with human mitochondrial ATP synthase components

  • Toxicity testing in mammalian cell lines

  • Structural comparison between bacterial and human homologs

  • Activity assays with site-directed mutants mimicking human protein

• Translational development strategies:

  • Liposomal delivery systems for ATP synthase inhibitors

  • Combination testing with established antibiotics

  • Resistance development monitoring through serial passage

  • Animal infection models to validate in vivo efficacy

These approaches leverage the structural and functional distinctiveness of bacterial ATP synthase compared to its mammalian counterpart. The essential nature of ATP synthesis for bacterial survival, combined with structural differences between bacterial and human ATP synthases, positions atpH and other ATP synthase components as promising targets for selective antimicrobial development.