Recombinant Pseudomonas aeruginosa ATP synthase protein I (atpI)

What is the structure and function of ATP synthase protein I (atpI) in Pseudomonas aeruginosa?

ATP synthase protein I (atpI) is a 126-amino acid membrane protein that serves as a component of the larger ATP synthase complex in Pseudomonas aeruginosa . The ATP synthase complex consists of two main sectors: the F1 sector (a soluble portion) and the Fo sector (membrane-bound portion). While the specific function of atpI in P. aeruginosa has not been fully characterized, it is likely involved in the assembly and stabilization of the ATP synthase complex.

The ATP synthase complex functions by utilizing the energy derived from a proton gradient to generate ATP. Protons cross the membrane through the Fo portion of the enzyme, establishing a proton-motive force. This energy causes rotation of two rotary motors: the c-ring in Fo and the associated subunits in F1, ultimately driving ATP synthesis .

Unlike eukaryotic ATP synthase, which contains regulatory proteins such as IF1 that have no prokaryotic counterparts, bacterial ATP synthase complexes like that in P. aeruginosa have a simpler regulatory system. This reflects the evolutionary conservation and specialization of ATP synthases across different domains of life .

How does the ATP synthase assembly process work in bacteria compared to eukaryotes?

The assembly of ATP synthase in bacteria differs from that in eukaryotes in several key aspects:

• In eukaryotes, ATP synthase assembly involves multiple factors and chaperones, including Factor B, which has no prokaryotic homologue .

• The eukaryotic assembly process involves three different modules: the c-ring, F1, and the Atp6p/Atp8p complex .

• For yeast ATP synthase, assembly involves two separate pathways (F1/Atp9p and Atp6p/Atp8p/2 stator subunits/Atp10p chaperone) that converge at the end stage .

• In bacteria like P. aeruginosa, the assembly process appears to be simpler, typically involving the assembly of the c-ring followed by binding of F1, the stator arm, and finally subunits a and related proteins .

• Eukaryotic ATP synthase assembly is translationally regulated to achieve a balanced output between nuclear-encoded and mitochondrially-encoded subunits .

What are the optimal methods for expressing and purifying recombinant P. aeruginosa atpI?

Expression and purification of recombinant P. aeruginosa atpI requires careful consideration of expression systems and purification strategies:

Expression Systems:

• E. coli expression system: This has been successfully used to produce recombinant full-length P. aeruginosa atpI (1-126aa) with an N-terminal His tag . E. coli BL21(DE3) or similar strains are typically used with pET-based expression vectors.

• Alternative expression systems: While E. coli is the most common system, other options include yeast, baculovirus, or mammalian cells, which might be advantageous for certain applications requiring specific post-translational modifications .

Purification Protocol:

• Cell lysis: Bacterial cells expressing atpI are typically lysed using sonication, French press, or detergent-based methods in the presence of protease inhibitors.

• Membrane fraction isolation: Since atpI is a membrane protein, the membrane fraction is usually isolated by ultracentrifugation following cell lysis.

• Solubilization: Membrane proteins require detergent solubilization. Common detergents include n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-glucopyranoside (OG), or digitonin.

• Affinity chromatography: His-tagged atpI can be purified using immobilized metal affinity chromatography (IMAC) with Ni-NTA or similar resins .

• Size exclusion chromatography: A final polishing step using size exclusion chromatography can remove aggregates and improve purity.

Storage Conditions:
The purified protein is typically stored as a lyophilized powder or in solution with glycerol (5-50% final concentration) at -20°C or -80°C to avoid repeated freeze-thaw cycles .

What experimental approaches can be used to study the role of atpI in ATP synthase assembly?

Several methodological approaches can be employed to investigate the role of atpI in ATP synthase assembly:

• Gene knockout/knockdown studies:

  • CRISPR-Cas9 or traditional homologous recombination to create atpI deletion mutants

  • Analysis of ATP synthase complex formation in the absence of atpI

  • Complementation with wild-type or mutant atpI to restore assembly

• Site-directed mutagenesis:

  • Introduction of point mutations in conserved residues

  • Analysis of the effects on ATP synthase assembly and function

  • Identification of critical residues for protein-protein interactions

• Protein-protein interaction studies:

  • Co-immunoprecipitation of atpI with other ATP synthase subunits

  • Yeast two-hybrid or bacterial two-hybrid assays

  • Surface plasmon resonance or microscale thermophoresis to measure binding affinities

  • Cross-linking mass spectrometry to identify interaction interfaces

• Structural studies:

  • Cryo-electron microscopy of the ATP synthase complex with and without atpI

  • NMR studies of atpI-subunit interactions

  • X-ray crystallography of atpI alone or in complex with binding partners

• In vivo assembly monitoring:

  • Pulse-chase experiments to track the incorporation of newly synthesized atpI into the ATP synthase complex

  • Fluorescently tagged subunits to visualize assembly in real-time

  • Blue native PAGE to analyze complex formation under different conditions

These approaches can provide insights into whether atpI serves as a scaffolding protein, a chaperone, or has a more direct role in ATP synthase function.

How does P. aeruginosa atpI contribute to bacterial pathogenesis and antibiotic resistance?

While direct evidence linking atpI to pathogenesis or antibiotic resistance is limited in the search results, several hypotheses and experimental approaches can be considered:

P. aeruginosa is recognized as a multidrug resistant pathogen with intrinsically advanced antibiotic resistance mechanisms and is associated with serious illnesses such as hospital-acquired infections including ventilator-associated pneumonia and sepsis syndromes .

Potential Contributions to Pathogenesis:

• Energy metabolism: As part of ATP synthase, atpI likely contributes to maintaining the energy status of the bacterium, which is crucial for virulence factor production and survival within the host.

• Adaptation to microenvironments: ATP synthesis capabilities may allow P. aeruginosa to adapt to different host microenvironments where oxygen availability and energy sources vary.

• Surface-associated ATP synthase: Similar to cancer cells, P. aeruginosa might potentially have ectopic ATP synthase on its surface, which could contribute to extracellular ATP generation affecting the host microenvironment .

Experimental Approaches:

• Virulence assessment: Compare the virulence of wild-type and atpI mutant strains in infection models.

• Antibiotic susceptibility testing: Determine whether atpI mutations affect susceptibility to different classes of antibiotics.

• Transcriptomics under infection conditions: Analyze atpI expression changes during infection or upon exposure to host defense mechanisms.

• ATP synthase inhibitor studies: Test whether specific inhibitors of ATP synthase affect P. aeruginosa virulence or antibiotic resistance.

• Metabolomics: Compare metabolic profiles of wild-type and atpI-deficient strains to understand the metabolic consequences of atpI disruption.

What are the optimal conditions for functional assays of recombinant P. aeruginosa atpI?

Functional characterization of recombinant P. aeruginosa atpI requires carefully designed assays under optimal conditions:

ATP Synthesis Assay Protocol:

• Reconstitution into proteoliposomes:

  • Purified recombinant ATP synthase complex containing atpI is incorporated into liposomes composed of a mixture of phosphatidylcholine and phosphatidic acid (9:1 ratio)

  • Reconstitution is performed via detergent dialysis or using polystyrene beads for detergent removal

• Establishment of proton gradient:

  • Proteoliposomes are prepared in acidic buffer (pH 5.0)

  • They are then diluted into alkaline buffer (pH 8.0) containing valinomycin and K+ to generate both a pH gradient and membrane potential

• ATP synthesis measurement:

  • Reaction is initiated by adding ADP (0.5 mM) and inorganic phosphate (10 mM)

  • Samples are taken at defined time points and ATP production is measured via luciferase-based ATP detection assay

ATP Hydrolysis Assay:

• Enzyme preparation:

  • Either purified enzyme in detergent or reconstituted into proteoliposomes

• Reaction conditions:

  • Buffer: 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2

  • ATP concentration: 1-5 mM

  • Temperature: 37°C (physiological relevance)

• Activity measurement:

  • ATPase activity measured by detecting released inorganic phosphate using malachite green assay

  • Alternative method: coupled enzyme assay with pyruvate kinase and lactate dehydrogenase

Control Experiments:

• Positive control: E. coli ATP synthase (well-characterized)

• Negative controls: Proteoliposomes without ATP synthase

• Inhibitor controls: Addition of specific inhibitors (e.g., DCCD, oligomycin) to confirm ATP synthase-specific activity

How can researchers identify potential inhibitors of P. aeruginosa atpI for antimicrobial development?

Given the importance of ATP synthase for bacterial survival and the multidrug resistance of P. aeruginosa , targeting atpI might represent a novel antimicrobial strategy:

High-throughput Screening Approaches:

• Biochemical assays:

  • ATP synthesis/hydrolysis assays in a 96/384-well format using purified ATP synthase containing atpI

  • Compounds that inhibit these activities are identified as primary hits

• Whole-cell screening:

  • Growth inhibition assays using wild-type P. aeruginosa and atpI overexpression strains

  • Compounds showing differential activity between these strains might target atpI

• Surface plasmon resonance (SPR) screening:

  • Immobilized atpI is used to screen for direct binding compounds

  • Binding kinetics and affinity can be determined

Structure-based Approaches:

• Virtual screening:

  • Using homology models or experimental structures of atpI

  • Molecular docking to identify compounds that bind to critical sites

  • Molecular dynamics simulations to assess binding stability

• Fragment-based drug discovery:

  • Screening small molecular fragments that bind to atpI

  • Growing or linking fragments to create more potent inhibitors

Validation and Optimization:

• Selectivity assessment:

  • Testing activity against human ATP synthase to ensure selectivity

  • Evaluation of activity against other bacterial ATP synthases

• Mechanism of action studies:

  • Confirming that inhibition occurs through interaction with atpI rather than other ATP synthase subunits

  • Site-directed mutagenesis to identify resistance mutations

• Pharmacokinetic optimization:

  • Modification of lead compounds to improve stability, solubility, and penetration into P. aeruginosa

What structural analysis techniques are most suitable for studying P. aeruginosa atpI?

Multiple structural biology techniques can be applied to study P. aeruginosa atpI, each with specific advantages:

Cryo-electron Microscopy (Cryo-EM):

• Advantages:

  • Can determine structures of membrane proteins without crystallization

  • Captures different conformational states

  • Can visualize atpI in the context of the entire ATP synthase complex

• Methodology:

  • Purified ATP synthase complex is flash-frozen on EM grids

  • Images are collected and processed to generate 3D reconstructions

  • Resolution can reach near-atomic levels (2-3 Å)

X-ray Crystallography:

• Advantages:

  • Potential for very high resolution (better than 2 Å)

  • Well-established phasing methods

• Challenges:

  • Crystallization of membrane proteins is difficult

  • May require detergents or lipidic cubic phase methods

  • May not capture dynamic aspects of atpI function

Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Advantages:

  • Provides dynamic information

  • Can work with membrane proteins in detergent micelles or nanodiscs

  • Useful for studying protein-protein interactions

• Limitations:

  • Size limitations (typically <30 kDa for detailed structure)

  • Requires isotope labeling (15N, 13C, 2H)

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

• Applications:

  • Mapping protein-protein interaction surfaces

  • Identifying conformational changes upon binding

  • Does not require crystallization

Computational Approaches:

• Homology modeling:

  • Using known structures of homologous proteins as templates

  • Particularly useful if experimental structures are unavailable

• Molecular dynamics simulations:

  • Investigating dynamics of atpI in membrane environments

  • Studying conformational changes during function

How can researchers distinguish between different conformational states of atpI during ATP synthesis?

Understanding the conformational changes of atpI during ATP synthesis is crucial for elucidating its function:

Single-molecule FRET:

• Methodology:

  • Introduction of fluorescent dye pairs at specific positions in atpI

  • Measurement of distance changes between dyes during ATP synthesis

  • Detection of different conformational states and transitions

• Key considerations:

  • Selection of labeling positions that don't disrupt function

  • Use of photobleaching-resistant fluorophores

  • Need for functional validation of labeled protein

Time-resolved cryo-EM:

• Approach:

  • Capturing ATP synthase complexes at different stages of the catalytic cycle

  • Trapping using different nucleotide analogs or inhibitors

  • Classification of particles into different conformational states

EPR spectroscopy:

• Methodology:

  • Site-directed spin labeling of atpI at selected positions

  • Continuous wave or pulsed EPR to measure distances between labels

  • Monitoring conformational changes under different conditions

Molecular dynamics simulations:

• Applications:

  • In silico modeling of atpI conformational changes

  • Testing hypotheses about motion during catalytic cycle

  • Identifying residues critical for conformational transitions

Cross-linking mass spectrometry:

• Approach:

  • Chemical cross-linking of atpI under different functional states

  • Mass spectrometric identification of cross-linked residues

  • Mapping distance constraints onto structural models

These methods can reveal how atpI might change conformation during the rotation of the c-ring and the associated conformational changes in the ATP synthase complex.

What are the challenges in studying protein-protein interactions between atpI and other ATP synthase subunits?

Investigating the interactions between atpI and other ATP synthase subunits presents several technical challenges:

Challenges:

• Membrane protein nature: The hydrophobic nature of atpI makes it difficult to study using conventional protein-protein interaction techniques.

• Complex stability: The ATP synthase complex may dissociate under conditions used for many interaction studies.

• Transient interactions: Some interactions may be dynamic or occur only during certain stages of assembly or function.

• Detergent interference: Detergents used to solubilize membrane proteins can interfere with protein-protein interactions.

Methodological Solutions:

• In situ approaches:

  • Chemical cross-linking followed by mass spectrometry

  • Proximity labeling methods (BioID, APEX) in living cells

  • FRET-based interaction studies in membranes

• Detergent-free systems:

  • Nanodiscs or liposomes to maintain membrane environment

  • Styrene-maleic acid lipid particles (SMALPs) to extract membrane protein complexes with their native lipid environment

• Split reporter systems:

  • Split GFP complementation

  • Bacterial two-hybrid systems adapted for membrane proteins

  • Protein-fragment complementation assays

• Quantitative binding assays:

  • Microscale thermophoresis with fluorescently labeled proteins

  • Bio-layer interferometry with immobilized proteins

  • Isothermal titration calorimetry optimized for membrane proteins

Each of these approaches has strengths and limitations, and a combination of methods is often necessary to fully characterize the interactions of atpI with other ATP synthase subunits.

How can researchers engineer recombinant P. aeruginosa atpI for enhanced structural studies?

Engineering recombinant atpI can significantly improve outcomes in structural biology studies:

Construct Design Strategies:

• Terminal modifications:

  • Addition of purification tags (His, Strep, FLAG) at positions that don't interfere with function

  • Inclusion of cleavable linkers between tags and atpI sequence

• Fusion proteins:

  • Fusion with crystallization chaperones (e.g., T4 lysozyme, BRIL)

  • Addition of fluorescent proteins for localization studies

  • MBP or GST fusions for improved solubility

• Truncation constructs:

  • Removal of disordered regions identified by prediction algorithms

  • Creation of minimal functional domains

• Surface engineering:

  • Introduction of surface mutations to enhance crystallizability

  • Reduction of surface entropy by replacing flexible side chains (Lys/Glu/Gln) with smaller residues (Ala)

Expression Optimization:

• Codon optimization: Adapting codon usage for the expression host

• Fusion partners: Using fusion proteins known to enhance expression (e.g., MBP, SUMO)

• Expression tags: Testing different combinations of N- and C-terminal tags

• Expression hosts: Screening multiple expression systems (E. coli, yeast, insect cells)

Stability Engineering:

• Disulfide bonds: Introduction of disulfide bonds to stabilize certain conformations

• Thermostabilizing mutations: Identification of mutations that enhance thermal stability

• Lipid environment optimization: Screening different lipids or detergents for optimal stability

A systematic approach testing multiple constructs in parallel often yields the best results for challenging membrane proteins like atpI.