Recombinant Campylobacter jejuni subsp. jejuni serotype O:23/36 ATP synthase subunit c (atpE)

What is the role of ATP synthase subunit c in Campylobacter jejuni energy metabolism?

ATP synthase subunit c forms the transmembrane c-ring within the F0 portion of the ATP synthase complex. In C. jejuni, this subunit is crucial for proton translocation across the bacterial membrane, directly coupling the proton motive force (pmf) to ATP synthesis. The c-ring rotates as protons pass through the membrane via a channel formed by subunits a and c, with proton binding to a conserved carboxyl residue in the middle of the membrane. This rotation drives conformational changes in the F1 catalytic domain, enabling ATP synthesis. In C. jejuni specifically, ATP generation via oxidative phosphorylation is essential for survival in microaerobic environments, such as the gastrointestinal tract of warm-blooded animals .

How does the structure of ATP synthase subunit c in C. jejuni compare to other bacterial species?

The ATP synthase subunit c of C. jejuni shares structural homology with other bacterial species but possesses unique features. Like other bacterial c subunits, it contains two transmembrane helices connected by a polar loop, but sequence alignments reveal species-specific variations. The key functional residue equivalent to Asp61 in E. coli or Glu59 in yeast is conserved in C. jejuni and is located in the middle of the C-terminal transmembrane helix . This residue is critical for proton translocation. The c-ring in C. jejuni likely contains 10-12 c subunits, though the exact stoichiometry differs between species and affects the bioenergetic efficiency of the ATP synthase complex.

What expression systems are most effective for producing recombinant C. jejuni ATP synthase subunit c?

Expression should be optimized through systematic testing of induction parameters:

• IPTG concentration: Start with range testing (0.1-1.0 mM)

• Induction temperature: Lower temperatures (16-25°C) often improve membrane protein folding

• Induction time: Extended periods (16-24 hours) at lower temperatures

• Media composition: Addition of glucose (0.5-1%) can reduce basal expression

• Consider auto-induction methods for higher biomass and protein yields

The incorporation of a periplasmic targeting sequence may improve proper folding and reduce toxicity associated with cytoplasmic accumulation of membrane proteins.

What purification strategies overcome the challenges associated with ATP synthase subunit c isolation?

Purifying recombinant ATP synthase subunit c presents unique challenges due to its hydrophobicity and membrane association. An effective purification workflow includes:

• Membrane Fraction Isolation:

  • Differential centrifugation following cell lysis (typically 100,000×g for 1 hour)

  • Careful washing of membrane pellets to remove peripheral proteins

• Solubilization:

  • Test multiple detergents (DDM, LDAO, C12E8) at various concentrations

  • Include stabilizing agents (glycerol 10%, specific lipids)

• Affinity Chromatography:

  • If using His-tagged constructs, use Ni-NTA or TALON resins

  • Include detergent in all buffers at concentrations above CMC

  • Consider on-column detergent exchange if needed

  • Use imidazole gradient elution to minimize non-specific binding

• Size Exclusion Chromatography:

  • Critical for separating monomeric from oligomeric forms

  • Can also remove detergent micelles and aggregates

• Special Considerations:

  • Addition of protease inhibitors is crucial to prevent degradation

  • The c-subunit tends to form SDS-resistant oligomers that may appear on SDS-PAGE

  • Consider native purification if functional studies are planned

Protein purity should be assessed by SDS-PAGE, and identity confirmed by mass spectrometry of intact protein and digested peptides .

How can researchers evaluate the proper folding and assembly of recombinant ATP synthase subunit c?

Verifying proper folding and assembly of recombinant ATP synthase subunit c requires multiple complementary approaches:

• Circular Dichroism (CD) Spectroscopy:

  • Confirms secondary structure elements

  • Expected high α-helical content (characteristic minima at 208 and 222 nm)

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Determines oligomeric state and homogeneity

  • Can distinguish between monomeric and ring-assembled forms

• Reconstitution into Liposomes:

  • Measures proton translocation activity

  • Can be assessed using pH-sensitive fluorescent dyes

• Binding Studies with Known Inhibitors:

  • Oligomycin, venturicidin, or DCCD should bind with expected affinities

  • Binding can be measured by isothermal titration calorimetry or fluorescence-based assays

• Mass Spectrometry:

  • Ion mobility mass spectrometry can assess tertiary and quaternary structure

  • Can identify post-translational modifications or unexpected truncations

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps solvent-accessible regions to confirm proper folding

  • Can identify regions involved in subunit interactions

Functional assays should also be performed to confirm biological activity, such as reconstitution with other ATP synthase subunits to measure ATP hydrolysis or synthesis activities.

What methods are most reliable for measuring ATP synthase activity in recombinant C. jejuni systems?

Several complementary approaches can be used to measure ATP synthase activity in recombinant C. jejuni systems:

• ATP Hydrolysis Assays:

  • Coupled enzyme assays (ATP hydrolysis coupled to NADH oxidation)

  • Pi-release assays using malachite green or molybdate

  • Luciferase-based ATP consumption assays

• ATP Synthesis Measurements:

  • Reconstituted proteoliposomes with artificially imposed proton gradient

  • Luciferase-based ATP detection

  • Real-time monitoring using pH-sensitive fluorescent dyes

• Proton Pumping Assays:

  • Acridine orange fluorescence quenching in proteoliposomes

  • ACMA (9-amino-6-chloro-2-methoxyacridine) fluorescence measurements

  • Voltage-sensitive dyes to monitor membrane potential changes

• Membrane Potential Measurements:

  • Fluorescent probe (DiSC3(5)) for measuring Δψ changes

  • Direct electrode measurements in reconstituted systems

  • Flow cytometry with appropriate membrane potential dyes

When working with the isolated c subunit, it's important to note that many functional assays require reconstitution with other ATP synthase components. Alternatively, the c subunit can be characterized by its binding to specific inhibitors and its ability to form oligomeric rings.

How do specific inhibitors interact with C. jejuni ATP synthase subunit c, and what insights do they provide about structure-function relationships?

Inhibitors targeting ATP synthase subunit c provide valuable insights into its structure-function relationships in C. jejuni:

• Oligomycin:

  • Binds at the interface of subunits a and c

  • Involves both the N- and C-terminal transmembrane helices of subunit c

  • In C. jejuni, as in other organisms, key residues in the middle of the membrane are likely involved in binding

  • Blocks proton translocation by interfering with the rotation of the c-ring

• Venturicidin:

  • Interacts with subunit c in the middle of the membrane

  • The binding region overlaps with that of oligomycin

  • Resistance mutations can be mapped to specific residues in the C-terminal helix

• DCCD (N,N'-dicyclohexylcarbodiimide):

  • Covalently modifies the key acidic residue involved in proton translocation

  • In C. jejuni, this would target the equivalent of E. coli Asp61

  • Provides direct evidence of the proton-binding site

• Other Inhibitors:

  • Efrapeptin targets the F1 domain but may provide insights into the coupled rotation mechanism

  • Aurovertin binds to the β subunit and inhibits ATPase activity uncompetitively

Inhibitor studies can reveal:

• The specific residues involved in proton translocation

• Structural differences between C. jejuni and other bacterial ATP synthases

• Potential antimicrobial targets specific to Campylobacter

Cross-resistance patterns between different inhibitors can map the three-dimensional arrangement of residues within the membrane-embedded portions of the protein.

What role does ATP synthase play in C. jejuni pathogenesis and host colonization?

ATP synthase is integral to C. jejuni pathogenesis and host colonization through several mechanisms:

• Energy Generation:

  • C. jejuni relies on oxidative phosphorylation for ATP production

  • ATP synthase couples the membrane potential to ATP synthesis

  • This energy is essential for various virulence mechanisms

• Membrane Potential Maintenance:

  • The proton motive force generated in part by ATP synthase is crucial for:

    • Flagellar rotation and motility

    • Nutrient transport

    • Protein secretion systems

  • Loss of membrane potential severely impairs colonization ability

• Adaptation to Different Environments:

  • ATP synthase activity helps C. jejuni adapt to varying oxygen tensions in the host

  • In the anaerobic gut lumen, formate or hydrogen serves as electron donors

  • Near epithelial cells, oxygen can be used as an electron acceptor

  • Both conditions require ATP synthase for energy conversion

• Connection to Motility:

  • Motility is essential for host colonization

  • ATP synthase contributes to the pmf needed for flagellar rotation

  • Experimental evolution shows that loss of motility leads to colonization defects

• Potential Role in Stress Responses:

  • ATP synthase may be involved in acid tolerance

  • Contributes to survival during transit through the stomach

  • May be regulated during oxidative stress

The critical role of ATP synthase in C. jejuni biology makes it a potential target for novel antimicrobial strategies against this foodborne pathogen.

What strategies can be used to create site-directed mutations in the atpE gene of C. jejuni?

Creating site-directed mutations in the C. jejuni atpE gene requires specialized approaches due to the organism's unique genetic characteristics:

• Shuttle Vector Systems:

  • E. coli/C. jejuni shuttle vectors (e.g., pRY111, pMEK91)

  • Include appropriate selection markers (chloramphenicol, kanamycin resistance)

  • Need Campylobacter-specific promoters for expression

• Allelic Exchange Methods:

  • Homologous recombination using suicide vectors

  • Double crossover selection using positive (antibiotic resistance) and negative (sacB) markers

  • Typically requires 500-1000 bp homology regions flanking the mutation site

• CRISPR-Cas9 Approaches:

  • Modified systems optimized for C. jejuni

  • Design sgRNAs targeting specific sites in atpE

  • Include repair templates carrying desired mutations

  • Increased efficiency compared to traditional methods

• Natural Transformation:

  • C. jejuni is naturally competent under specific conditions

  • PCR products with flanking homology regions can be used

  • Efficiency can be enhanced by using methylation-deficient DNA

• Considerations for atpE Mutations:

  • Essential gene mutations may require complementation strategies

  • Conditional systems can be used for lethal mutations

  • Single-copy chromosomal expression from alternative loci may be preferable to plasmid-based expression

For validation of mutations, sequencing should be combined with phenotypic analysis, including growth rate measurements, ATP synthesis assays, and proton translocation studies to assess the functional impact of the mutations.

How can researchers analyze the effects of atpE mutations on C. jejuni growth, survival, and pathogenesis?

A comprehensive analysis of atpE mutations requires multiple experimental approaches:

• Growth and Survival Characterization:

  • Growth curves under various conditions (microaerobic, aerobic, anaerobic)

  • Survival assays in stress conditions (acid, bile salts, oxidative stress)

  • Determination of ATP content using luciferase-based assays

  • Measurement of membrane potential using DiSC3(5) or other potentiometric dyes

• Cellular Energetics Assessment:

  • Oxygen consumption rates using respirometry

  • Measurement of intracellular pH

  • Determination of proton motive force components (Δψ and ΔpH)

  • Comparison of ATP production via oxidative phosphorylation vs. substrate-level phosphorylation

• Motility Analysis:

  • Soft agar motility assays

  • Video microscopy to determine swimming patterns and velocities

  • Analysis of flagellar assembly by electron microscopy

  • Correlation between membrane potential and motility

• In Vitro Virulence Assays:

  • Adhesion and invasion assays using intestinal epithelial cell lines

  • Intracellular survival assays

  • Resistance to host defense mechanisms (complement, antimicrobial peptides)

  • Biofilm formation capacity

• In Vivo Models:

  • Colonization studies in appropriate animal models (chickens, mice)

  • Competition assays between wild-type and mutant strains

  • Disease progression in susceptible models (e.g., C57BL/6 IL-10 –/– mice)

  • Recovery and analysis of bacteria after passage through hosts

• Transcriptomic and Proteomic Analysis:

  • RNA-seq to identify compensatory changes in gene expression

  • Proteomics to determine effects on protein levels and modifications

  • Metabolomics to identify shifts in metabolic pathways

These approaches provide a comprehensive understanding of how atpE mutations affect multiple aspects of C. jejuni physiology and pathogenesis.

What insights can comparative genomics provide about the evolution of ATP synthase subunit c in different Campylobacter species and strains?

Comparative genomics offers significant insights into ATP synthase subunit c evolution:

• Sequence Conservation Analysis:

  • Identification of highly conserved residues across Campylobacter species

  • Mapping of variable regions that may relate to niche adaptation

  • Selection pressure analysis (dN/dS ratios) to identify residues under positive selection

  • Correlation between sequence variations and ecological niches

• Genomic Context Examination:

  • Comparison of the atp operon organization across Campylobacter species

  • Identification of regulatory elements and their conservation

  • Analysis of horizontal gene transfer events affecting ATP synthase genes

  • Genome reduction patterns in relation to host adaptation

• Phylogenetic Analysis:

  • Construction of phylogenetic trees based on atpE sequences

  • Comparison with whole-genome phylogenies to identify discordant patterns

  • Molecular clock analyses to estimate divergence times

  • Correlation with host specificity and pathogenicity

• Structural Predictions:

  • Homology modeling of ATP synthase subunit c across species

  • Identification of species-specific structural features

  • Prediction of functional consequences of observed variations

  • Analysis of c-ring stoichiometry differences between species

• Comparison with Free-Living Relatives:

  • Analysis of ATP synthase evolution in Campylobacter compared to Sulfurospirillum

  • Identification of adaptations related to host-association vs. free-living lifestyle

  • Assessment of genome reduction patterns affecting energy metabolism genes

These comparative approaches can reveal how ATP synthase has evolved in Campylobacter during adaptation to various hosts and environmental niches, potentially identifying species-specific features that could be targeted for diagnostic or therapeutic purposes.

What advanced structural techniques can be applied to study the c-ring assembly of C. jejuni ATP synthase?

Several cutting-edge structural techniques can elucidate the c-ring assembly of C. jejuni ATP synthase:

• Cryo-Electron Microscopy (Cryo-EM):

  • Single-particle analysis for high-resolution structure determination

  • Can resolve the complete ATP synthase complex including the c-ring

  • Tomography for visualizing the complex in native membrane environments

  • Sample preparation requires detergent solubilization or nanodiscs

• X-ray Crystallography:

  • Requires crystallization of purified c-rings

  • Can provide atomic-level resolution of the c-subunit arrangement

  • Typically requires lipid cubic phase or vapor diffusion methods

  • May need specific antibody fragments as crystallization chaperones

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Solution NMR for dynamics studies of the isolated c-subunit

  • Solid-state NMR for studying the assembled c-ring in membranes

  • Magic-angle spinning techniques for high-resolution spectra

  • Isotopic labeling (15N, 13C, 2H) required for detailed studies

• Atomic Force Microscopy (AFM):

  • Visualizes c-rings in native-like membrane environments

  • Can provide topographical information and mechanical properties

  • High-speed AFM can capture conformational changes during function

  • Force spectroscopy to measure interactions between subunits

• Mass Spectrometry-Based Approaches:

  • Native mass spectrometry to determine oligomeric state

  • Ion mobility MS to characterize conformational states

  • Cross-linking MS to map subunit interfaces

  • HDX-MS to identify dynamic regions and binding interfaces

• Computational Methods:

  • Molecular dynamics simulations of the c-ring in membrane environment

  • Coarse-grained simulations for long-timescale dynamics

  • Homology modeling based on existing c-ring structures

  • QM/MM simulations for proton transfer mechanisms

Integrating multiple structural techniques provides complementary information and overcomes limitations of individual methods.

How can researchers investigate the proton translocation mechanism in C. jejuni ATP synthase subunit c?

Investigating the proton translocation mechanism requires specialized techniques:

• Site-Directed Mutagenesis Combined with Functional Assays:

  • Mutation of the key proton-binding residue (equivalent to E. coli Asp61)

  • Alteration of surrounding residues to modify pKa or accessibility

  • Measurement of proton pumping using pH-sensitive fluorescent dyes

  • Correlation of mutations with ATP synthesis/hydrolysis rates

• pH-Dependent Spectroscopic Studies:

  • FTIR difference spectroscopy to monitor protonation states

  • Solid-state NMR to detect pH-dependent chemical shifts

  • Raman spectroscopy to identify protonation-dependent conformational changes

  • Time-resolved studies to capture transient intermediates

• Electrophysiological Approaches:

  • Patch-clamp recording of proteoliposomes containing ATP synthase

  • Planar lipid bilayer recordings to measure proton currents

  • Single-molecule FRET to monitor conformational changes during proton translocation

  • Correlation of proton flux with c-ring rotation

• Chemical Probes and Modifications:

  • DCCD labeling to identify proton-binding sites

  • pH-dependent cross-linking to map conformational changes

  • Cysteine scanning mutagenesis with thiol-reactive probes

  • Environmental-sensitive fluorescent labels to detect local changes

• Molecular Dynamics Simulations:

  • QM/MM simulations of proton transfer events

  • pKa calculations for the key acidic residue in different environments

  • Free energy calculations for proton binding and release

  • Simulation of water dynamics in the proton translocation pathway

• Comparison of Proton/Ion Specificity:

  • Testing Na+ vs. H+ specificity of C. jejuni ATP synthase

  • Identification of residues determining ion selectivity

  • Measurement of competing ion effects on function

These approaches can elucidate the unique features of proton translocation in C. jejuni ATP synthase and identify potential differences from model systems like E. coli.

What computational approaches are most valuable for predicting structure-function relationships in C. jejuni ATP synthase subunit c?

Computational approaches provide valuable insights into structure-function relationships:

• Homology Modeling and Threading:

  • Generation of structural models based on existing c-ring structures

  • Assessment of model quality using validation tools

  • Comparison across multiple templates to identify conserved features

  • Integration with experimental constraints from cross-linking or spectroscopic data

• Molecular Dynamics (MD) Simulations:

  • All-atom simulations in explicit membrane environments

  • Analysis of protein stability and conformational dynamics

  • Identification of water molecules and their pathways

  • Characterization of lipid-protein interactions specific to C. jejuni

• Enhanced Sampling Methods:

  • Umbrella sampling to calculate free energy profiles for proton transfer

  • Metadynamics to identify energy barriers in conformational transitions

  • Replica exchange simulations to improve conformational sampling

  • Transition path sampling to identify rare events in proton translocation

• Quantum Mechanical Calculations:

  • QM/MM studies of proton transfer reactions

  • Calculation of pKa values for key residues

  • Analysis of electronic structure during proton binding/release

  • Correlation of calculated energetics with experimental rate measurements

• Network Analysis and Machine Learning:

  • Identification of evolutionary coupled residues using statistical coupling analysis

  • Prediction of functional hotspots using random forest or neural network approaches

  • Classification of mutations as benign or deleterious

  • Integration of genomic data to identify co-evolving positions

• Docking and Virtual Screening:

  • In silico screening for novel inhibitors targeting C. jejuni-specific features

  • Modeling of known inhibitor binding modes (oligomycin, venturicidin)

  • Calculation of binding free energies for inhibitor-protein complexes

  • Identification of species-specific binding pockets

These computational approaches, especially when integrated with experimental data, provide a deeper understanding of ATP synthase mechanism and guide experimental design for further studies.

How can structural differences in ATP synthase subunit c between C. jejuni and human mitochondria be exploited for selective inhibitor design?

Developing selective inhibitors requires identifying and targeting structural differences:

• Comparative Structural Analysis:

  • Sequence alignment and structural comparison between C. jejuni and human ATP synthase

  • Identification of amino acid differences in the proton channel and inhibitor binding regions

  • Analysis of c-ring stoichiometry differences (typically 8 subunits in mammals vs. 10-12 in bacteria)

  • Mapping of species-specific surface features and binding pockets

• Key Exploitable Differences:

  • C. jejuni likely contains a different arrangement of residues around the conserved proton-binding site

  • The interface between subunits a and c has species-specific features

  • The lipid-binding regions differ due to membrane composition differences

  • The central cavity of the c-ring may present bacterial-specific binding sites

• Rational Inhibitor Design Strategies:

  • Structure-based design targeting C. jejuni-specific pockets

  • Modification of existing inhibitors (oligomycin, venturicidin) to increase selectivity

  • Fragment-based approaches to identify novel chemotypes

  • Peptide-based inhibitors mimicking natural inhibitory sequences

• Computational Approaches:

  • Virtual screening against C. jejuni ATP synthase models

  • Molecular dynamics simulations to identify transient pockets

  • Free energy calculations to predict binding selectivity

  • Pharmacophore modeling based on known inhibitors

• Experimental Validation:

  • Biochemical assays comparing inhibition of bacterial vs. mammalian ATP synthase

  • Cell-based assays measuring selective toxicity

  • Resistance mutation analysis to confirm binding sites

  • Structural studies of inhibitor-bound complexes

The goal is to develop compounds that potently inhibit C. jejuni ATP synthase while showing minimal activity against human mitochondrial ATP synthase, thereby reducing toxicity concerns.

What evidence supports the potential of ATP synthase as an antimicrobial target in C. jejuni?

Multiple lines of evidence support ATP synthase as an antimicrobial target:

• Essentiality for Growth and Survival:

  • C. jejuni relies heavily on oxidative phosphorylation for energy production

  • The membrane potential generated is crucial for multiple cellular functions

  • Unlike some bacteria, C. jejuni has limited metabolic flexibility to compensate for ATP synthase inhibition

• Role in Pathogenesis:

  • ATP synthase function is linked to motility, a key virulence determinant

  • The membrane potential is essential for host colonization

  • Energy generation is required for resistance to host defense mechanisms

• Precedent in Other Organisms:

  • Bedaquiline, targeting mycobacterial ATP synthase, is approved for treating tuberculosis

  • Various natural products (oligomycin, venturicidin) inhibit bacterial ATP synthases

  • The essential nature of ATP synthase is conserved across diverse bacterial pathogens

• Structural Uniqueness:

  • Differences in c-ring stoichiometry between bacterial and mammalian ATP synthases

  • Species-specific residues in inhibitor binding regions

  • Distinct regulatory mechanisms in bacterial vs. mammalian enzymes

• Limited Resistance Development:

  • Multiple subunits involved in function means multiple mutations may be needed for resistance

  • Essential nature limits tolerance for mutations

  • Membrane environment constraints restrict permissible variations

• Synergistic Potential:

  • ATP synthase inhibitors may synergize with existing antibiotics

  • Combination therapy approaches could reduce resistance development

  • Energy depletion may sensitize bacteria to other stressors

These factors collectively support the potential of ATP synthase inhibitors as a novel therapeutic approach against C. jejuni infections.

How might ATP synthase inhibitors affect C. jejuni biofilm formation and antibiotic resistance?

ATP synthase inhibitors could impact biofilm formation and antibiotic resistance through several mechanisms:

Understanding these relationships could guide the development of combination therapies that target both ATP synthesis and other cellular processes to effectively combat C. jejuni infections.

What are the latest technological advances for studying ATP synthase function in vivo?

Recent technological innovations have revolutionized the study of ATP synthase function in vivo:

• Genetically Encoded Biosensors:

  • FRET-based ATP sensors allow real-time visualization of ATP levels in living cells

  • pH-responsive fluorescent proteins to monitor local pH changes around ATP synthase

  • Membrane potential sensors with improved sensitivity and temporal resolution

  • Targeted sensors that localize specifically to ATP synthase in the membrane

• Advanced Microscopy Techniques:

  • Super-resolution microscopy (STORM, PALM) to visualize individual ATP synthase complexes

  • Light-sheet microscopy for 3D imaging with reduced photodamage

  • Single-molecule tracking to monitor ATP synthase dynamics in live cells

  • Correlative light and electron microscopy to link function with ultrastructure

• Cryo-Electron Tomography:

  • Visualization of ATP synthase in its native cellular environment

  • Subtomogram averaging to obtain structural information in situ

  • Focused ion beam milling to prepare samples from intact bacterial cells

  • Correlative cryo-fluorescence microscopy for targeted tomography

• Optogenetic Approaches:

  • Light-controllable proton pumps to manipulate pmf in real-time

  • Optogenetic control of ATP synthase expression or assembly

  • Photoswitchable inhibitors for temporally precise inhibition

  • Light-induced conformational changes in engineered ATP synthase variants

• In Vivo NMR and Metabolomics:

  • Real-time 31P-NMR to monitor ATP synthesis rates

  • Metabolic flux analysis using stable isotope labeling

  • Spatial metabolomics to map ATP distribution within bacterial populations

  • Integration with transcriptomics and proteomics for systems-level understanding

These technologies enable researchers to study ATP synthase with unprecedented spatial and temporal resolution in living bacterial cells, providing insights into its function in native contexts.

How does ATP synthase function integrate with other aspects of C. jejuni metabolism under different environmental conditions?

ATP synthase integrates with multiple metabolic systems that vary with environmental conditions:

• Oxygen-Dependent Metabolic Shifts:

  • Under microaerobic conditions, C. jejuni uses a branched electron transport chain

  • ATP synthase activity coordinates with respiratory complexes

  • Oxygen availability influences the expression of donor complexes (formate dehydrogenase, hydrogenase)

  • Transition to anaerobic respiration requires ATP synthase adjustment to altered pmf

• Carbon Source Utilization:

  • Different carbon sources affect the NADH/FADH2 ratio, influencing electron transport

  • ATP synthase activity must adapt to changing proton pumping stoichiometries

  • Carbon limitation may trigger energy conservation mechanisms affecting ATP synthase regulation

  • Integration with substrate-level phosphorylation pathways via AckA

• Response to Environmental Stressors:

  • pH stress requires ATP synthase adaptation to maintain internal pH

  • Osmotic stress affects membrane properties and potentially c-ring rotation

  • Temperature changes influence membrane fluidity and ATP synthase efficiency

  • Oxidative stress may damage ATP synthase components, requiring repair mechanisms

• Connection to Virulence Mechanisms:

  • ATP synthase provides energy for flagellar motility

  • Integration with chemotaxis systems for directed movement

  • Energy supply for invasion and intracellular survival

  • Coordinate regulation with virulence gene expression

• Metabolic Network Modeling Approaches:

  • Flux balance analysis to predict ATP synthase contribution under different conditions

  • Integration of transcriptomic and proteomic data into metabolic models

  • Dynamic modeling of ATP production in response to environmental shifts

  • Identification of synthetic lethal interactions with ATP synthase

Understanding these integrated networks can reveal how C. jejuni maintains energy homeostasis across diverse host environments and identify potential vulnerabilities for therapeutic targeting.

What potential roles might ATP synthase play in C. jejuni stress responses and antibiotic tolerance?

ATP synthase likely plays multifaceted roles in stress responses and antibiotic tolerance:

• Acid Stress Response:

  • ATP synthase may contribute to cytoplasmic pH homeostasis

  • Reverse operation (ATP hydrolysis driving proton export) under extreme acid stress

  • Altered expression or regulation in response to gastric transit

  • Integration with other acid resistance mechanisms

• Oxidative Stress Management:

  • ATP supply for antioxidant defense systems

  • Potential role in maintaining redox balance

  • Possible target of oxidative damage requiring repair

  • Connection to iron homeostasis systems

• Antibiotic Tolerance Mechanisms:

  • Energy requirement for adaptive resistance responses

  • Role in persister cell formation through energy depletion

  • ATP-dependent efflux pump function

  • Membrane potential contribution affecting antibiotic uptake

• Nutrient Limitation Responses:

  • Adaptation to carbon or phosphate starvation

  • Integration with stringent response pathways

  • Role in metabolic dormancy under severe starvation

  • Energy conservation mechanisms during nutrient transition periods

• Biofilm-Associated Stress Tolerance:

  • Energy requirements for biofilm matrix production

  • Metabolic heterogeneity within biofilm populations

  • Adaptation to oxygen gradients in biofilms

  • Integration with quorum sensing systems

• Temperature Stress Adaptation:

  • Cold shock response and protein folding energy requirements

  • Heat shock response and chaperone system integration

  • Membrane fluidity adaptation affecting c-ring rotation

  • Seasonal variation in host environments

Research in these areas could identify how C. jejuni modulates ATP synthase function to survive in diverse host environments and potentially reveal new approaches to combat antimicrobial resistance in this pathogen.

What mass spectrometry approaches are most effective for characterizing recombinant C. jejuni ATP synthase subunit c?

Multiple mass spectrometry approaches provide complementary information for characterization:

• Intact Protein Analysis:

  • ESI-MS for accurate molecular weight determination

  • Detection of post-translational modifications or processing events

  • Confirmation of proper signal peptide cleavage

  • Assessment of sample homogeneity

• Ion Mobility MS:

  • Separation of protein conformers

  • Determination of collision cross-section values

  • Analysis of oligomeric state distribution

  • Characterization of protein-lipid complexes

• Bottom-up Proteomics:

  • Peptide mapping after enzymatic digestion

  • Sequence coverage confirmation

  • Identification of modified residues

  • Quantitative analysis using labeled reference peptides

• Top-down Proteomics:

  • Fragmentation of intact protein for sequence verification

  • Localization of modifications without prior digestion

  • Detection of unexpected processing events

  • Analysis of proteoforms

• Native MS:

  • Analysis of oligomeric assemblies (c-rings)

  • Binding studies with lipids or inhibitors

  • Determination of subunit stoichiometry

  • Stability assessment under different conditions

• Hydrogen-Deuterium Exchange MS:

  • Mapping of solvent-accessible regions

  • Detection of conformational changes upon ligand binding

  • Identification of protein-protein interaction interfaces

  • Comparison of dynamics between wild-type and mutant proteins

• Metal Analysis by ICP-MS:

  • Quantification of any associated metal ions

  • Determination of metal:protein stoichiometry

  • Analysis of metal binding affinity

  • Assessment of sample purity

These approaches should be combined for comprehensive characterization of recombinant ATP synthase subunit c.

What are the most reliable methods for assessing purity and homogeneity of recombinant ATP synthase subunit c preparations?

A multi-technique approach ensures reliable assessment of purity and homogeneity:

• SDS-PAGE Analysis:

  • Multiple staining methods (Coomassie, silver, fluorescent)

  • Densitometry for quantitative purity assessment

  • Western blotting for specific detection

  • Assessment of SDS-resistant oligomers common in c-subunits

• Size Exclusion Chromatography:

  • Detection of aggregates and oligomeric species

  • Multi-angle light scattering (SEC-MALS) for molecular weight determination

  • Monitoring at multiple wavelengths to detect non-protein contaminants

  • Analytical ultracentrifugation for detailed oligomeric state analysis

• Mass Spectrometry:

  • High-resolution MS for detection of minor species

  • Intact mass analysis for post-translational modifications

  • Native MS for oligomeric state assessment

  • MALDI-TOF for rapid quality control

• Spectroscopic Methods:

  • UV-Vis spectroscopy for protein concentration and contaminant detection

  • Circular dichroism for secondary structure confirmation

  • Fluorescence spectroscopy for tertiary structure assessment

  • FTIR for assessment of secondary structure in membrane environment

• Chromatographic Techniques:

  • Reversed-phase HPLC for hydrophobic variant detection

  • Ion exchange chromatography for charge variant analysis

  • Hydrophobic interaction chromatography for conformational variants

  • Affinity chromatography with specific ligands

• Functional Homogeneity Assessment:

  • Binding assays with known inhibitors

  • Reconstitution experiments for functional testing

  • Thermal stability assays (DSF, nanoDSC)

  • Activity correlations with different fractions

A specific challenge for ATP synthase subunit c is its high hydrophobicity, which may cause anomalous migration on SDS-PAGE and elution behavior in chromatography. Therefore, multiple orthogonal techniques should be employed for comprehensive quality assessment.

How can researchers ensure proper folding and membrane insertion of recombinant ATP synthase subunit c?

Ensuring proper folding and membrane insertion requires specialized approaches:

• Structural Characterization:

  • Circular dichroism to confirm high α-helical content

  • FTIR spectroscopy to assess secondary structure in membrane environments

  • Fluorescence spectroscopy to monitor tertiary structure

  • Limited proteolysis to identify protected regions

• Detergent and Lipid Screening:

  • Systematic testing of different detergents for solubilization

  • Reconstitution in various lipid compositions

  • Native PAGE analysis in different detergent systems

  • Thermal stability measurements in different environments

• Functional Validation:

  • Binding studies with known inhibitors (oligomycin, DCCD)

  • Assembly into c-rings in vitro

  • Proton translocation assays in proteoliposomes

  • Integration with other ATP synthase components

• Membrane Insertion Assays:

  • Protease protection assays to confirm topology

  • Fluorescence quenching to assess membrane integration

  • Chemical labeling of accessible residues

  • Freeze-fracture electron microscopy to visualize membrane incorporation

• Expression System Considerations:

  • Use of specialized expression hosts for membrane proteins

  • Co-expression with chaperones

  • Inclusion of proper signal sequences

  • Slow induction at lower temperatures

• Computational Validation:

  • Molecular dynamics simulations to assess stability in membrane

  • Hydrophobicity analysis to predict membrane-spanning regions

  • Comparison with known structures through homology modeling

  • Energy minimization to detect unfavorable conformations

The combination of these approaches provides confidence in the proper folding and membrane insertion of recombinant ATP synthase subunit c, which is critical for structural and functional studies.