Recombinant Roseobacter denitrificans ATP synthase subunit c (atpE)

What is the function of ATP synthase subunit c in Roseobacter denitrificans?

ATP synthase subunit c (atpE) in R. denitrificans forms part of the oligomeric c-ring in F₀F₁-ATP synthase. This essential membrane protein participates in the rotational mechanism that couples proton translocation through F₀ to ATP synthesis. The c-ring rotates relative to the a-subunit, driven by the proton motive force across the membrane. Key carboxyl residues in the c-subunits are involved in proton uptake and release, facilitating energy transduction necessary for ATP production in this marine bacterium .

How does the structure of R. denitrificans ATP synthase subunit c compare to other bacterial homologs?

R. denitrificans atpE shares the fundamental structural features common to bacterial ATP synthase c-subunits, including:

• A hairpin-like structure with two transmembrane α-helices

• A conserved carboxyl residue (glutamic or aspartic acid) essential for proton binding

• Oligomerization capacity to form the c-ring (typically containing 8-15 c-subunits)

While the core structure is conserved, R. denitrificans shows adaptations consistent with its marine environment, possibly including salt-bridge formations and surface charge distributions that maintain functionality in high-salt conditions .

What expression systems are most suitable for producing recombinant R. denitrificans atpE?

For recombinant expression of R. denitrificans atpE, E. coli-based systems have proven effective, though with important considerations:

When using E. coli, optimization of induction conditions (temperature, IPTG concentration) is critical. For R. denitrificans atpE, lower induction temperatures (16-20°C) and moderate IPTG concentrations (0.1-0.5 mM) generally yield better results. The use of specialized media with controlled salt concentrations is recommended to accommodate the protein's marine origin while maintaining antibiotic effectiveness for selection .

What are the optimal conditions for site-directed mutagenesis of R. denitrificans atpE?

Site-directed mutagenesis of R. denitrificans atpE requires careful optimization:

• Template preparation: Use high-quality plasmid DNA (>50 ng/μL) with verified sequence

• Primer design:

  • Forward and reverse primers should contain the desired mutation centrally positioned

  • Primers should be 25-45 nucleotides long with GC content 40-60%

  • Terminal G or C bases help anchoring

  • Calculate melting temperature (Tm) >78°C for the mutagenic primers

• PCR conditions:

  • Use high-fidelity DNA polymerase (e.g., Pfu Ultra or Q5)

  • Initial denaturation: 95°C for 2 minutes

  • 16-18 cycles of: 95°C for 30s, 55°C for 1 min, 72°C for 1 min/kb

  • Final extension: 72°C for 10 minutes

• DpnI digestion: 1 hour at 37°C to remove template DNA

• Transformation: Use highly competent cells (>10⁸ transformants/μg)

The conservation of key functional residues (e.g., the proton-binding glutamate) should be considered when designing mutations. Comparative analysis with other bacterial atpE sequences can guide target selection for mutagenesis .

What purification strategies yield the highest purity and activity for recombinant R. denitrificans atpE?

Purification of recombinant R. denitrificans atpE requires specialized approaches due to its hydrophobic nature:

• Membrane preparation:

  • Harvest cells and resuspend in buffer (typically 50 mM Tris-HCl pH 8.0, 100 mM NaCl)

  • Disrupt cells via French press or sonication (3-5 cycles, 30s on/30s off)

  • Remove unbroken cells by centrifugation (10,000×g, 20 min)

  • Isolate membranes by ultracentrifugation (150,000×g, 1 hour)

• Solubilization:

  • Resuspend membrane fraction in solubilization buffer

  • Add detergent (1-2% n-dodecyl-β-D-maltoside or 1% digitonin)

  • Incubate with gentle agitation (4°C, 1-2 hours)

  • Remove insoluble material by ultracentrifugation (150,000×g, 30 min)

• Chromatography sequence:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged protein

  • Size exclusion chromatography to remove aggregates

  • Optional ion exchange chromatography for final polishing

• Quality control:

  • SDS-PAGE with silver staining (>95% purity)

  • Western blotting with anti-His or specific anti-atpE antibodies

  • Dynamic light scattering to assess homogeneity

Maintaining 0.05% detergent throughout all purification steps is critical to prevent protein aggregation. Consider adding 10% glycerol to all buffers to enhance protein stability .

How can I accurately determine the oligomeric state of recombinant R. denitrificans atpE?

Determining the oligomeric state of R. denitrificans atpE requires complementary approaches:

• Blue Native PAGE:

  • Sample preparation with mild detergents to maintain native interactions

  • Gradient gels (4-16%) allow separation of different oligomeric states

  • Reference proteins of known molecular weight for calibration

• Analytical ultracentrifugation:

  • Sedimentation velocity experiments at 40,000-50,000 rpm

  • Analysis using SEDFIT software for continuous c(s) distribution

  • Multiple detergent concentrations to verify detergent-independent associations

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS):

  • Accurate molecular weight determination independent of shape

  • Correction for detergent contribution to signal

  • Determination of protein:detergent ratio

• Negative stain electron microscopy:

  • Direct visualization of ring structure

  • Statistical analysis of particle dimensions

  • 2D class averaging to enhance structural details

This combinatorial approach provides robust verification of the oligomeric state. Based on studies with other bacteria, the c-ring in R. denitrificans likely contains 10-15 subunits, forming a ring structure essential for rotational function in ATP synthesis .

How do mutations in R. denitrificans atpE affect proton translocation and ATP synthesis activities?

Mutations in R. denitrificans atpE can significantly impact ATP synthase function, as demonstrated in studies with related systems:

• Mutations of the key carboxyl residue:

  • Conservative substitutions (e.g., Glu→Asp) reduce activity but may not eliminate it

  • Non-conservative substitutions (e.g., Glu→Gln) typically abolish proton translocation and ATP synthesis

  • These findings suggest the critical importance of the carboxyl group's protonation/deprotonation capacity

• Functional consequences:

  • Single mutations reduce ATP synthesis activity (typically to 30-40% of wild-type levels)

  • Double mutations further decrease activity, with greater separation between mutation sites causing more severe effects

  • These patterns indicate cooperation among c-subunits during rotation

• Mechanistic implications:

  • Prolonged proton uptake times in mutated c-subunits

  • Sharing of waiting time for protonation between adjacent c-subunits

  • Disruption of cooperative interactions as mutations are spaced further apart

Experimental approaches to study these effects include ATP synthesis assays using inverted membrane vesicles, proton pump activity measurements, and molecular dynamics simulations that can reveal atomic-level changes in proton transfer pathways .

What methods can detect conformational changes in R. denitrificans atpE during the catalytic cycle?

Detecting conformational changes in R. denitrificans atpE during its catalytic cycle requires sophisticated biophysical approaches:

• Site-directed spin labeling with electron paramagnetic resonance (EPR):

  • Introduction of cysteine residues at strategic positions

  • Labeling with paramagnetic probes (e.g., MTSL)

  • Distance measurements between labeled sites during different catalytic states

  • Requires careful selection of labeling positions to avoid functional disruption

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Measures solvent accessibility changes during the catalytic cycle

  • Can be performed in near-native membrane environments

  • Provides regional (peptide-level) resolution of conformational dynamics

  • Requires optimization for membrane proteins in detergent micelles

• Single-molecule Förster resonance energy transfer (smFRET):

  • Site-specific labeling with donor-acceptor fluorophore pairs

  • Real-time observation of distance changes during rotation

  • Can capture transient intermediates missed by ensemble methods

  • Technical challenges include maintaining protein functionality after labeling

• Time-resolved cryo-electron microscopy:

  • Capture of structural snapshots during the catalytic cycle

  • Visualization of c-ring rotation relative to other subunits

  • Requires advanced sample preparation and image processing

  • Most informative when performed on intact F₀F₁ complexes

These methods provide complementary information about the conformational dynamics of atpE during proton translocation and c-ring rotation, helping to elucidate the molecular mechanism of energy conversion .

How does the antibiotic resistance profile of R. denitrificans influence genetic manipulation of its atpE gene?

R. denitrificans exhibits intrinsic resistance to several antibiotics, which significantly impacts genetic manipulation strategies for its atpE gene:

The presence of β-lactamase genes and aminoglycoside phosphotransferase genes in R. denitrificans explains its resistance profile. For genetic manipulation:

• Vector selection: Choose plasmids with spectinomycin resistance markers for optimal selection

• Media composition: Use half-strength Marine Broth (hMB) rather than full-strength MB to enhance antibiotic efficacy

• Transformation protocols: Higher antibiotic concentrations and longer expression times after transformation

• Verification methods: Include multiple confirmation steps (PCR, sequencing) to verify successful genetic modifications

This antibiotic resistance profile necessitates careful planning for any genetic work involving R. denitrificans atpE, including recombinant expression systems and mutagenesis studies .

What are the most effective assays for measuring ATP synthase activity in recombinant R. denitrificans atpE preparations?

Assessing ATP synthase activity in recombinant R. denitrificans atpE systems requires specialized assays that can be adapted from established protocols:

• ATP synthesis activity assay:

  • Preparation of inverted membrane vesicles through French press or sonication

  • Energization of vesicles with NADH or succinate to generate proton gradient

  • Measurement of ATP production using luciferase-based luminescence assays

  • Controls should include protonophore (CCCP) to confirm proton-gradient dependence

• ATP hydrolysis activity measurement:

  • Spectrophotometric coupled enzyme assay (PK-LDH) linking ATP hydrolysis to NADH oxidation

  • Direct measurement of inorganic phosphate release (malachite green assay)

  • Inhibitor controls (oligomycin, DCCD) to confirm specificity

  • Compare activity before and after DCCD treatment to quantify c-subunit-specific effects

• Proton pumping assays:

  • Monitoring pH changes using ACMA fluorescence quenching

  • Measurement of membrane potential using potential-sensitive dyes (e.g., oxonol VI)

  • Quantification of H⁺/ATP ratio through simultaneous measurement of proton translocation and ATP hydrolysis

• Single-molecule rotation assays:

  • Immobilization of F₁ domain on glass surfaces

  • Attachment of fluorescent markers to c-ring

  • Real-time observation of rotation under ATP hydrolysis conditions

  • Analysis of step size, rotation rate, and effects of mutations

These methods enable comprehensive characterization of both ATP synthesis/hydrolysis activities and the coupling efficiency between proton translocation and catalytic function .

How can molecular dynamics simulations complement experimental studies of R. denitrificans atpE?

Molecular dynamics (MD) simulations provide valuable insights into R. denitrificans atpE structure and function that complement experimental approaches:

• Proton transfer pathway analysis:

  • Simulation of protonation/deprotonation events at key residues

  • Identification of water molecules in proton transfer pathways

  • Calculation of energy barriers for proton movement

  • Prediction of effects of specific mutations on proton transfer

• Conformational dynamics investigation:

  • Characterization of c-ring stability and flexibility in membrane environment

  • Analysis of interactions between adjacent c-subunits

  • Identification of cooperative movements during rotation

  • Simulation of interaction interfaces between c-ring and a-subunit

• Simulation methodology:

  • System preparation with explicit membrane and solvent

  • Equilibration (typically 10-50 ns) followed by production runs (100+ ns)

  • Enhanced sampling techniques (metadynamics, umbrella sampling) for rare events

  • Analysis of hydrogen bond networks, salt bridges, and water distribution

• Integration with experimental data:

  • Validation of simulation results against biochemical measurements

  • Use of simulations to interpret experimental observations

  • Design of new experiments based on simulation predictions

  • Iterative refinement of both computational and experimental approaches

Recent proton transfer-coupled MD simulations have successfully reproduced experimental findings, such as the cooperative behavior of c-subunits and the effects of mutations on waiting times for proton uptake. These computational approaches are particularly valuable for understanding atomic-level details that are challenging to observe experimentally .

What structural comparison techniques reveal functional differences between R. denitrificans atpE and homologs from other bacteria?

Structural comparison of R. denitrificans atpE with homologs from other bacteria provides insights into adaptation and functional specialization:

• Sequence-based analyses:

  • Multiple sequence alignment to identify conserved and variable regions

  • Calculation of conservation scores for each position

  • Phylogenetic analysis to relate sequence divergence to evolutionary relationships

  • Correlation of sequence features with habitat (marine vs. terrestrial, thermophilic vs. mesophilic)

• Homology modeling and structural comparison:

  • Construction of R. denitrificans atpE model based on available structures

  • Superposition analysis to quantify structural deviations

  • Comparison of electrostatic surface potentials

  • Analysis of packing interactions within the c-ring

• Functional site analysis:

  • Comparison of proton-binding sites (pKa values, local environment)

  • Analysis of interfaces with other ATP synthase subunits

  • Identification of species-specific structural features

  • Correlation of structural differences with functional properties

• Experimental validation approaches:

  • Creation of chimeric proteins combining segments from different species

  • Site-directed mutagenesis to introduce signature residues from other species

  • Functional assays to measure effects of structural alterations

  • Cross-species complementation studies

Key differences likely include adaptations to salt concentration in the marine environment, optimizations for the specific bioenergetic requirements of R. denitrificans, and variations in c-ring stoichiometry that affect the H⁺/ATP ratio .

What are common problems in recombinant expression of R. denitrificans atpE and how can they be resolved?

Recombinant expression of R. denitrificans atpE presents several challenges that require systematic troubleshooting:

When standard troubleshooting fails, consider these advanced approaches:

• Cell-free expression systems that bypass cellular toxicity issues

• Expression as separate transmembrane segments followed by reconstitution

• Co-expression with other F₀ subunits to promote proper assembly

• Nanodiscs or amphipol systems for detergent-free membrane protein stabilization .

How can R. denitrificans atpE be effectively reconstituted into liposomes for functional studies?

Reconstitution of R. denitrificans atpE into liposomes requires careful optimization for functional studies:

• Liposome preparation:

  • Selection of lipid composition: typically a mixture of POPC, POPE, and POPG (7:2:1) with 10% cholesterol

  • For marine bacteria-derived proteins, consider adding specific bacterial lipids (2-5%)

  • Preparation methods: extrusion through polycarbonate filters (100-400 nm) or reverse-phase evaporation

  • Buffer composition should mimic physiological conditions (pH 7.5-8.0, 100-300 mM NaCl)

• Protein incorporation protocols:

  • Detergent-mediated reconstitution using controlled detergent removal

  • Detergent removal methods: Bio-Beads SM2, dialysis, or gel filtration

  • Protein:lipid ratio optimization (typically 1:50 to 1:200 w/w)

  • Orientation control using pH gradients during reconstitution

• Functional verification:

  • Proton pumping assays using pH-sensitive fluorescent dyes

  • ATP synthesis measurements upon establishment of proton gradient

  • Patch-clamp electrophysiology for direct measurement of proton conductance

  • Freeze-fracture electron microscopy to verify incorporation and distribution

• Critical parameters:

  • Temperature control during reconstitution (4-25°C)

  • Detergent removal rate (slower is generally better)

  • Final detergent concentration below CMC

  • Proteoliposome storage conditions (-80°C with cryoprotectants like trehalose)

Successful reconstitution enables detailed biophysical and functional characterization of R. denitrificans atpE under controlled conditions, allowing precise measurement of proton translocation properties .

What are the latest advances in studying interactions between R. denitrificans atpE and potential inhibitors?

Recent methodological advances have enhanced our ability to study interactions between R. denitrificans atpE and potential inhibitors:

• In silico screening approaches:

  • Structure-based virtual screening using homology models

  • Molecular docking with flexible protein conformations

  • Pharmacophore modeling based on known inhibitors

  • MD simulation-based binding free energy calculations

• Binding affinity determination:

  • Microscale thermophoresis (MST) for membrane protein-ligand interactions

  • Surface plasmon resonance (SPR) with captured proteoliposomes

  • Isothermal titration calorimetry (ITC) with detergent-solubilized protein

  • Fluorescence-based thermal shift assays adapted for membrane proteins

• Structural characterization of inhibitor binding:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map binding interfaces

  • Solid-state NMR studies of inhibitor-bound reconstituted samples

  • Cross-linking mass spectrometry to identify binding sites

  • Cryo-EM of inhibitor-bound complexes

• Functional inhibition assays:

  • ATP synthesis inhibition in inverted membrane vesicles

  • Proton translocation blocking measured by fluorescence quenching

  • Growth inhibition studies in bacterial cultures

  • Synergistic effects with other antibiotics

Computational studies have identified several compounds with potential inhibitory activity against bacterial ATP synthase c-subunits, showing binding energies ranging from -8.69 to -8.44 kcal/mol. These compounds typically interact with the proton-binding site or interfere with the rotation mechanism of the c-ring .

How does the marine environment influence the structure and function of R. denitrificans atpE?

The marine environment exerts unique selective pressures on R. denitrificans atpE structure and function:

• Adaptation to high salt concentration:

  • Modified surface charge distribution to maintain stability in high ionic strength

  • Altered hydrophobic/hydrophilic balance in transmembrane regions

  • Specialized ion coordination sites to manage cation interactions

  • These adaptations can be identified through comparative analysis with terrestrial bacterial homologs

• Bioenergetic considerations:

  • Optimization for operation under marine pH conditions (typically pH 7.8-8.2)

  • Adjustments in proton-binding residue pKa values

  • Adaptations to variable oxygen availability in marine environments

  • Potential modifications to accommodate alternative coupling ions

• Structural specializations:

  • c-ring stoichiometry adaptations affecting the H⁺/ATP ratio

  • Interface modifications for interaction with marine-adapted a-subunit

  • Lipid-protein interactions optimized for marine bacterial membranes

  • Potential unique structural elements for stability in cold marine environments

• Experimental approaches to study environmental adaptation:

  • Functional studies across varying salt concentrations and pH values

  • Lipid composition effects on activity and stability

  • Pressure-dependent studies to mimic depth variations

  • Temperature-activity profiles to understand thermal adaptation

Understanding these adaptations provides insights into how essential bioenergetic machinery evolves to function in specialized ecological niches .

What cooperative mechanisms exist between R. denitrificans atpE subunits during ATP synthesis?

Cooperation among c-subunits in R. denitrificans ATP synthase is critical for efficient energy conversion:

• Fundamental cooperative mechanisms:

  • Sharing of waiting time for proton uptake between adjacent c-subunits

  • Coordinated conformational changes during c-ring rotation

  • Electrostatic interactions between neighboring subunits affecting protonation states

  • These mechanisms enable smooth, continuous rotation rather than discrete jumps

• Experimental evidence from related systems:

  • ATP synthesis activity decreases with increasing distance between mutated sites

  • Molecular dynamics simulations show shared proton uptake timing between subunits

  • Two to three deprotonated carboxyl residues typically face the a-subunit simultaneously

  • These patterns suggest that 2-3 c-subunits cooperate at the a/c interface during rotation

• Structural basis for cooperation:

  • Intersubunit hydrogen bonding networks

  • Transmembrane helix packing interactions

  • Water molecule coordination between subunits

  • Electrostatic networks spanning multiple subunits

• Functional consequences:

  • Enhanced efficiency of proton translocation

  • Smoother energy conversion with reduced energy barriers

  • Increased robustness against single-site perturbations

  • Optimized coupling between proton flow and mechanical rotation

Understanding these cooperative mechanisms is essential for developing a complete model of ATP synthase function and for designing potential inhibitors that might disrupt this cooperation .

How can structural information about R. denitrificans atpE inform drug discovery against related pathogenic bacteria?

Structural insights from R. denitrificans atpE can accelerate drug discovery against related pathogenic bacteria:

• Comparative structural analysis:

  • Identification of conserved features between R. denitrificans and pathogenic species

  • Mapping of species-specific differences that could be exploited for selectivity

  • Analysis of binding pocket conservation for inhibitor design

  • These comparisons help identify promising drug targets shared across species

• Rational drug design approaches:

  • Structure-based design of inhibitors targeting conserved functional sites

  • Fragment-based drug discovery using the c-subunit structure

  • Design of molecules that disrupt c-ring assembly or rotation

  • Virtual screening against R. denitrificans models to identify initial hits

• Selectivity considerations:

  • Targeting sequence and structural differences between bacterial and human ATP synthase

  • Exploitation of differences in c-ring stoichiometry between species

  • Focus on inhibitor binding sites present in bacteria but absent in mammals

  • Design of compounds that preferentially accumulate in bacterial membranes

• Methodological pipeline:

  • Initial screening against recombinant R. denitrificans atpE

  • Counter-screening against human ATP synthase for selectivity

  • Optimization of lead compounds for improved pharmacokinetics

  • Testing in whole-cell systems and animal models

Recent studies identified several compounds with binding energies in the range of -8.44 to -8.69 kcal/mol against mycobacterial ATP synthase c-subunit. Similar approaches could be applied using R. denitrificans atpE as a model system, potentially leading to new antibiotics against resistant pathogens .