Recombinant Idiomarina loihiensis ATP synthase subunit b (atpF), partial

What is the structural and functional role of ATP synthase subunit b (atpF) in Idiomarina loihiensis?

ATP synthase subunit b (atpF) in Idiomarina loihiensis functions as a critical component of the F-type ATP synthase complex (F₁F₀), specifically in the membrane-embedded F₀ sector. The protein forms part of the peripheral stalk that connects the catalytic F₁ domain to the membrane-embedded F₀ domain, providing structural stability to the complex while allowing for rotational catalysis. This structural support is essential for maintaining the proper orientation of the complex during ATP synthesis or hydrolysis. In bacterial systems like Idiomarina loihiensis, the b subunit typically exists as a dimer (b₂) and helps anchor the α₃β₃ hexamer to the membrane sector, enabling the enzyme to harness the proton motive force for ATP production .

How can researchers verify the identity and integrity of recombinant Idiomarina loihiensis atpF protein?

Verification of recombinant Idiomarina loihiensis atpF protein can be accomplished through several complementary methods:

• SDS-PAGE analysis: Confirm the expected molecular weight and >85% purity as indicated in product specifications .

• Western blotting: Use antibodies specific to either the atpF protein or the His-tag (if present).

• Mass spectrometry: Perform peptide mass fingerprinting to confirm the amino acid sequence.

• N-terminal sequencing: Verify the first 5-10 amino acids to confirm proper expression.

• Functional assays: Test the protein's ability to interact with other ATP synthase subunits using pull-down assays.

For optimal results, researchers should use at least two orthogonal methods for verification, with SDS-PAGE being the minimum standard as referenced in the product specifications .

What expression systems are available for Idiomarina loihiensis atpF production and their comparative advantages?

The choice of expression system should be guided by the specific research objectives. For basic structural studies, E. coli expression may be sufficient, while for complex functional studies examining interactions with other ATP synthase components, mammalian or baculovirus systems might be preferable .

How does the structure of Idiomarina loihiensis atpF compare with atpF homologs in other bacterial species?

While specific structural information for Idiomarina loihiensis atpF is limited, comparative analysis with better-characterized bacterial ATP synthase b subunits reveals several important features:

• Domain organization: Like other bacterial b subunits, I. loihiensis atpF likely contains a membrane-anchoring N-terminal domain and a predominantly alpha-helical C-terminal domain that extends into the cytoplasm.

• Dimerization interface: The C-terminal region typically forms a right-handed coiled-coil structure that enables dimerization, critical for forming the peripheral stalk.

• Species-specific adaptations: Based on studies of ATP synthases from other extremophiles, I. loihiensis atpF may contain adaptations related to its marine environment, potentially including salt-bridge forming residues that provide stability under varying ionic conditions.

Recent structural studies of ATP synthase from the photosynthetic bacterium Chloroflexus aurantiacus revealed a unique architecture with "a pair of peripheral stalks connect to the CaF₁ head through a dimer of δ-subunits, and associate with two membrane-embedded a-subunits" . This arrangement might have parallels in I. loihiensis, particularly in how the b subunit interfaces with other components of the complex.

What functional assays can be used to investigate the recombinant atpF in the context of ATP synthase activity?

Several sophisticated assays can be employed to investigate recombinant atpF functionality:

• Reconstitution assays: Incorporate purified recombinant atpF into proteoliposomes along with other ATP synthase subunits to measure ATP synthesis or hydrolysis rates. This approach has been successfully demonstrated with recombinant ATP synthase components from A. baumannii, where "purified A. baumannii F₁-ATPase (AbF₁-ATPase) composed of subunits α₃:β₃:γ:ε showed latent ATP hydrolysis" .

• FRET-based interaction studies: Label recombinant atpF and potential interaction partners with FRET donor/acceptor pairs to measure binding dynamics and conformational changes in real-time.

• ATP hydrolysis inhibition assays: Measure how varying concentrations of recombinant atpF affect the ATPase activity of partially assembled F₁ complexes.

• Crosslinking studies: Use chemical crosslinkers to capture interactions between atpF and other ATP synthase subunits, followed by mass spectrometry analysis to identify interaction interfaces.

• Hydrogen-deuterium exchange mass spectrometry: Map structural dynamics and solvent accessibility changes when atpF interacts with other subunits.

These approaches can be complemented by computational modeling to predict interaction surfaces and guide experimental design .

How can site-directed mutagenesis of Idiomarina loihiensis atpF inform structure-function relationships?

Site-directed mutagenesis provides a powerful approach to dissect the functional domains of atpF. Based on structural insights from related ATP synthases, researchers can target several key regions:

Following mutagenesis, functional impact can be assessed through reconstitution experiments similar to those described for A. baumannii F₁-ATPase, where "mutational studies of single amino acid substitutions within Abε or its interacting subunits β and γ, respectively, as well as C-terminal truncated mutants of Abε, provided a detailed picture of Abε's main element for the self-inhibition mechanism of ATP hydrolysis" .

What role might atpF play in the adaptation of Idiomarina loihiensis to its extreme marine environment?

Idiomarina loihiensis was initially isolated from the Lōiʻhi Seamount near Hawaii, an environment characterized by high pressure, variable temperatures, and unique ionic composition. The atpF protein likely plays crucial roles in environmental adaptation:

• Pressure adaptation: The structure of atpF may contain specific amino acid compositions that maintain proper folding and flexibility under high hydrostatic pressure conditions.

• Salt tolerance: As a halophilic organism, I. loihiensis likely has adaptations in membrane proteins like atpF to function optimally in high salt environments, potentially including an increased proportion of acidic amino acids on the protein surface.

• Energy efficiency: The ATP synthase complex, including atpF, may be optimized for energy conservation under nutrient-limited conditions typical of deep-sea environments.

• Thermal stability: Although not an extreme thermophile, I. loihiensis inhabits regions with hydrothermal activity, suggesting potential adaptations in atpF that contribute to thermal stability of the ATP synthase complex.

Comparative studies with atpF from non-extremophilic bacteria could highlight these adaptive features and potentially inform biotechnological applications requiring protein stability under extreme conditions .

What purification strategies yield the highest purity and functional integrity for recombinant atpF protein?

Optimal purification of recombinant Idiomarina loihiensis atpF requires a multi-step approach that preserves both purity and native structure:

• Initial capture: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA or TALON resins provides efficient initial purification.

• Intermediate purification: Ion exchange chromatography (typically anion exchange at pH 8.0) can separate the target protein from contaminants with different charge properties.

• Polishing step: Size exclusion chromatography separates monomeric from aggregated forms and removes remaining impurities.

• Detergent considerations: If purifying full-length atpF including its membrane domain, appropriate detergents (such as DDM, LDAO, or Brij-35) must be included throughout to maintain native structure.

• Buffer optimization: A Tris/PBS-based buffer with 6% trehalose at pH 8.0 has been successfully used for storage of related ATP synthase components from I. loihiensis .

The target purity should exceed 90% as determined by SDS-PAGE, similar to that achieved for other ATP synthase components from I. loihiensis . After purification, proper storage is critical - lyophilization or storage at -20°C/-80°C with the addition of 5-50% glycerol is recommended to maintain protein stability during long-term storage .

How can researchers reconstitute functional ATP synthase complexes using recombinant atpF?

Reconstitution of functional ATP synthase complexes incorporating recombinant atpF requires a systematic approach:

• Co-expression strategy: Express multiple ATP synthase subunits simultaneously in a suitable host system. This approach has been successful for generating A. baumannii F₁-ATPase composed of subunits α₃:β₃:γ:ε .

• Sequential assembly: Alternatively, purify individual subunits separately and assemble them in a controlled, step-wise manner:

  • Begin with the formation of subcomplexes (e.g., F₁ sector)

  • Incorporate membrane subunits including atpF into liposomes

  • Combine the soluble and membrane components

• Proteoliposome preparation:

  • Select appropriate lipids (typically E. coli polar lipids or a defined mixture of POPC:POPE:cardiolipin)

  • Control protein:lipid ratio (typically 1:50 to 1:100 w/w)

  • Ensure proper orientation of the complex (inside-out vesicles for ATP synthesis activity measurement)

• Functional validation:

  • ATP synthesis activity: Measure ATP production upon generation of a proton gradient

  • ATP hydrolysis activity: Assess if the complex exhibits expected regulatory properties, such as the "latent ATP hydrolysis" observed in A. baumannii F₁-ATPase

  • Proton pumping: Monitor pH changes or fluorescent probes to confirm proton translocation

• Cryo-EM structural analysis: Once functional complexes are obtained, structural integrity can be verified through cryo-electron microscopy, as demonstrated for the ATP synthase from Chloroflexus aurantiacus .

What techniques can effectively measure interactions between atpF and other ATP synthase subunits?

Several complementary techniques can quantitatively characterize the interactions between atpF and other ATP synthase components:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified atpF on a sensor chip

  • Flow solutions containing other ATP synthase subunits over the surface

  • Measure association and dissociation kinetics in real-time

  • Determine binding affinities (KD values)

• Isothermal Titration Calorimetry (ITC):

  • Directly measure thermodynamic parameters of binding (ΔH, ΔS, ΔG)

  • Determine stoichiometry of interactions

  • No immobilization or labeling required

• Microscale Thermophoresis (MST):

  • Label one interaction partner with a fluorescent dye

  • Measure changes in thermophoretic mobility upon binding

  • Requires small sample volumes and tolerates detergents

• Co-immunoprecipitation with quantitative mass spectrometry:

  • Use antibodies against atpF to pull down interaction complexes

  • Identify binding partners by mass spectrometry

  • Quantify relative affinities using SILAC or TMT labeling approaches

• NMR spectroscopy for structural characterization:

  • Map interaction interfaces at atomic resolution

  • Similar to the NMR solution structure studies performed for A. baumannii ε subunit, which revealed "interaction of its N-terminal β-barrel and C-terminal ɑ-hairpin domain"

These methods can help elucidate the specific residues involved in interactions and the strength of binding between atpF and other components of the ATP synthase complex.

How does the inhibition of ATP synthase affect bacterial metabolism and potential applications in antimicrobial research?

Inhibition of ATP synthase, including through targeting the atpF subunit, has profound effects on bacterial metabolism with implications for antimicrobial development:

• Metabolic consequences of ATP synthase inhibition:

  • Disruption of energy production leading to ATP depletion

  • Collapse of proton motive force across the membrane

  • Altered cellular redox state

  • Activation of stress response pathways

• Antimicrobial potential:

  • Research has shown that "inhibition of the ATP synthase sensitizes S. aureus towards polymyxins"

  • ATP synthase inhibition can eliminate intrinsic resistance to certain antibiotics

  • Combination therapy approaches may be particularly effective

• Experimental approaches to study atpF as an antimicrobial target:

  • Generate knockdown or conditional mutants of atpF to assess viability

  • Screen for small molecules that specifically disrupt atpF interactions

  • Employ ATP synthase inhibitors like oligomycin A alongside other antimicrobials to assess synergistic effects

• Species-specific considerations:

  • Different bacterial species show varying dependence on ATP synthase

  • Respiratory pathogens like A. baumannii may be particularly susceptible to ATP synthase targeting

  • Assess effects in both aerobic and anaerobic conditions

• Potential applications beyond antimicrobials:

  • Biotechnological applications requiring controlled energy production

  • Environmental applications targeting specific bacterial populations

  • Research tools for understanding bacterial bioenergetics

These approaches build on findings that "inhibition of the ATP synthase sensitizes S. aureus to this group of compounds [polymyxins]" and could potentially "enable the use of polymyxins against S. aureus and other Gram-positive pathogens" .

What strategies can address poor expression or solubility of recombinant Idiomarina loihiensis atpF?

Poor expression or solubility of recombinant atpF can be addressed through systematic optimization:

• Expression system modifications:

  • Try different host strains (BL21(DE3), C41(DE3), C43(DE3) for E. coli systems)

  • Test various induction conditions (temperature, inducer concentration, duration)

  • Use specialized expression vectors with solubility-enhancing tags (SUMO, MBP, TrxA)

• Construct optimization:

  • Express truncated versions lacking the hydrophobic N-terminal domain

  • Create chimeric constructs with well-expressed homologs

  • Optimize codon usage for the expression host

• Solubilization strategies:

  • Screen multiple detergents for membrane domain solubilization (DDM, LDAO, Brij-35)

  • Test detergent:protein ratios systematically

  • Consider alternative solubilization agents like SMALPs or nanodiscs

• Purification modifications:

  • Include stabilizing additives (glycerol, trehalose, specific lipids)

  • Optimize buffer conditions (pH, salt concentration, reducing agents)

  • Purify at reduced temperatures (4°C throughout)

• Co-expression approaches:

  • Co-express with natural binding partners (other ATP synthase subunits)

  • Include molecular chaperones (GroEL/ES, DnaK/J)

These strategies can be implemented sequentially or in combination to overcome expression and solubility challenges that are common with membrane-associated proteins like atpF.

How can researchers interpret structural data when investigating ATP synthase subunit interactions?

Interpreting structural data for ATP synthase subunit interactions requires consideration of several key factors:

• Membrane protein crystallography limitations:

  • Crystal structures may not capture the natural membrane environment

  • Detergents can distort native interactions

  • Resolution may be insufficient for water molecule or ion positions

• Cryo-EM considerations:

  • Different rotational states must be classified correctly

  • Flexibility of peripheral stalks may result in lower local resolution

  • Amphipathic regions may adopt non-native conformations in detergent

• Integrating multiple structural techniques:

  • Combine X-ray crystallography, cryo-EM, NMR, and SAXS data

  • Use crosslinking mass spectrometry to validate interaction surfaces

  • Apply molecular dynamics simulations to explore dynamic aspects

• Structure-function correlation:

  • Compare structural features with functional assays

  • Assess if observed structural states correspond to biochemically characterized states

  • Validate key interaction residues through mutagenesis

• Comparative analysis across species:

  • Use structures from related organisms to identify conserved interaction motifs

  • Consider evolutionary adaptations when interpreting species-specific features

  • The recent structure of ATP synthase from Chloroflexus aurantiacus provides valuable comparative data, revealing "a previously unrecognized architecture of ATP synthases"

By carefully integrating multiple structural approaches with functional data, researchers can develop robust models of how atpF interacts with other ATP synthase components in the native context.

What considerations are important when designing experiments to study the role of atpF in ATP synthase assembly?

Designing experiments to study atpF's role in ATP synthase assembly requires careful consideration of:

• Temporal aspects of assembly:

  • Develop pulse-chase experiments to track assembly intermediates

  • Use inducible expression systems to control timing of subunit availability

  • Consider co-translational vs. post-translational assembly events

• Spatial organization:

  • Investigate membrane targeting and insertion mechanisms

  • Examine the role of special membrane domains in assembly

  • Study interactions with assembly factors or chaperones

• Experimental approaches:

  • Create fluorescently tagged atpF to visualize localization and assembly in vivo

  • Develop in vitro assembly systems with purified components

  • Use chemical crosslinking to capture transient assembly intermediates

  • Apply native gel electrophoresis to identify stable subcomplexes

• Genetic approaches:

  • Generate conditional knockdowns or depletion strains

  • Create fusion proteins that allow inducible dimerization or oligomerization

  • Implement CRISPR interference for precise temporal control

• Control experiments:

  • Include proper controls for tag interference with assembly

  • Validate that experimental conditions don't introduce artifacts

  • Confirm that observed effects are specific to atpF and not general stress responses

These methodological considerations will help researchers design robust experiments to elucidate the specific role of atpF in the complex process of ATP synthase assembly.

What emerging technologies might advance our understanding of Idiomarina loihiensis ATP synthase structure and function?

Several cutting-edge technologies hold promise for advancing our understanding of I. loihiensis ATP synthase:

• Cryo-electron tomography (cryo-ET):

  • Study ATP synthase organization in native membrane environments

  • Observe supramolecular arrangements and potential oligomeric states

  • Capture different conformational states during the catalytic cycle

• Single-molecule techniques:

  • FRET-based approaches to measure subunit dynamics during rotation

  • Optical or magnetic tweezers to directly measure torque generation

  • Single-molecule force spectroscopy to probe mechanical stability

• Advanced computational methods:

  • AlphaFold or RoseTTAFold for prediction of complex structures

  • Molecular dynamics simulations incorporating lipid environments

  • Machine learning approaches to identify structural patterns across species

• Time-resolved structural methods:

  • Time-resolved cryo-EM to capture transient states

  • Serial crystallography at X-ray free electron lasers (XFELs)

  • Integrative structural biology combining multiple time-resolved techniques

• In-cell structural biology:

  • Cryo-FIB/SEM to visualize ATP synthase in its native cellular context

  • In-cell NMR to monitor structural changes in living cells

  • Proximity labeling approaches to map the ATP synthase interactome

These technologies could help address unresolved questions about ATP synthase function, particularly in understanding how the unique properties of I. loihiensis ATP synthase relate to its environmental adaptations.

How might comparative studies between different bacterial ATP synthases inform evolutionary adaptations?

Comparative studies of ATP synthases can reveal important evolutionary insights:

• Phylogenetic analysis of ATP synthase components:

  • Trace the evolutionary history of atpF and related subunits

  • Identify lineage-specific adaptations in extremophiles vs. mesophiles

  • Map conservation patterns to functional domains

• Structural comparisons across species:

  • Analyze ATP synthases from organisms in diverse environments

  • Identify structural adaptations for different energy sources

  • Compare I. loihiensis ATP synthase with those from other marine bacteria and extremophiles

• Functional adaptations to environmental pressures:

  • Examine coupling efficiency differences across species

  • Compare regulation mechanisms in different metabolic contexts

  • Study how proton binding sites have evolved for different pH optima

• Research approaches:

  • Generate chimeric ATP synthases with subunits from different species

  • Perform directed evolution experiments under defined selective pressures

  • Use ancestral sequence reconstruction to resurrect and characterize evolutionary intermediates

• Specific examples from literature:

  • The unique architecture of ATP synthase from the photosynthetic bacterium Chloroflexus aurantiacus with "two a-subunits that are asymmetrically positioned" provides an evolutionary perspective on adaptation to photosynthetic lifestyles

  • Findings from A. baumannii showing "a previously unrecognized architecture of ATP synthases" highlight the diversity of structural solutions to similar functional requirements

Such comparative approaches could reveal how I. loihiensis ATP synthase has adapted to its specific environmental niche and provide insights into the evolutionary diversification of this essential molecular machine.

What potential biotechnological applications might emerge from research on recombinant Idiomarina loihiensis ATP synthase components?

Research on I. loihiensis ATP synthase components could lead to several biotechnological applications:

• Bioenergy applications:

  • Development of ATP synthase-based biobatteries

  • Engineering more efficient molecular motors based on ATP synthase design principles

  • Creation of artificial energy converting membranes

• Antimicrobial development:

  • Design of specific inhibitors targeting bacterial ATP synthases

  • Sensitization strategies to enhance effectiveness of existing antibiotics

  • Based on findings that "inhibition of the ATP synthase eliminates the intrinsic resistance of Staphylococcus aureus to polymyxins"

• Protein engineering:

  • Stabilized membrane proteins for structural studies

  • Engineered protein scaffolds based on ATP synthase architecture

  • Design of novel molecular machines incorporating ATP synthase principles

• Biosensing technologies:

  • ATP synthase-based biosensors for environmental monitoring

  • Detection systems for proton gradient disruptors

  • Nanoscale pH sensors utilizing ATP synthase components

• Bionanotechnology:

  • Self-assembling nanostructures based on ATP synthase architecture

  • Molecular rotary motors for nanomechanical devices

  • Template designs for synthetic biology applications