Recombinant Maricaulis maris ATP synthase subunit b 2 (atpF2)

What is Maricaulis maris ATP synthase subunit b 2 and what role does it play in cellular energetics?

Maricaulis maris ATP synthase subunit b 2 (atpF2) is a component of the F-type ATP synthase complex, specifically part of the membrane-embedded F₀ sector. This protein plays a critical role in the structural organization of the ATP synthase complex, connecting the F₀ and F₁ domains and participating in the proton translocation pathway necessary for ATP synthesis. The complete protein consists of 183 amino acids and is encoded by the atpF2 gene (Mmar10_2204 locus) .

Unlike the catalytic subunits, subunit b 2 serves primarily as a structural component that facilitates the mechanical coupling between proton translocation through the membrane and the conformational changes required for ATP synthesis. The protein contributes to maintaining the structural integrity of the complex during rotational catalysis.

How should researchers optimally store and handle recombinant Maricaulis maris ATP synthase subunit b 2 for experimental use?

For optimal experimental outcomes when working with recombinant Maricaulis maris ATP synthase subunit b 2, researchers should follow these evidence-based handling protocols:

• Storage conditions: Store the protein at -20°C for routine use, or at -80°C for extended storage periods. The protein is typically supplied in a Tris-based buffer containing 50% glycerol optimized for stability .

• Avoid repeated freeze-thaw cycles: These can significantly compromise protein integrity. Instead, prepare small working aliquots during initial thawing .

• Working aliquots can be stored at 4°C for up to one week to minimize degradation from repeated temperature changes .

• When designing experiments, consider that the protein's activity may be affected by buffer composition, pH, and the presence of divalent cations such as Mg²⁺, which is essential for ATP synthase function.

• For functional studies, reconstitution into liposomes or nanodiscs may be necessary to preserve the native conformation of this membrane-associated protein.

How does ATP synthase subunit b 2 differ from other subunits in the F-type ATP synthase complex?

The F-type ATP synthase complex consists of multiple subunits with distinct functions. Subunit b 2 differs from other subunits in several key aspects:

Unlike the catalytic subunits that bind nucleotides and undergo conformational changes during catalysis, subunit b 2 serves as part of the stator, preventing the F₁ sector from rotating with the central stalk during ATP synthesis.

What methodological approaches are recommended for studying interactions between ATP synthase subunit b 2 and other components of the complex?

For investigating interactions between ATP synthase subunit b 2 and other components of the ATP synthase complex, researchers should consider these methodological approaches:

• Cross-linking studies coupled with mass spectrometry: This approach can identify interaction interfaces between subunit b 2 and neighboring subunits. Use photo-activatable or chemical cross-linkers followed by digestion and MS/MS analysis to map interaction sites.

• Cryo-electron microscopy (Cryo-EM): Recently used successfully to resolve structures of mycobacterial ATP synthases , this technique can capture the entire complex in different conformational states, revealing how subunit b 2 interfaces with other components.

• FRET-based assays: By labeling subunit b 2 and potential interaction partners with appropriate fluorophores, researchers can monitor real-time interactions and conformational changes during ATP synthesis/hydrolysis.

• Site-directed mutagenesis: Systematic mutation of conserved residues in subunit b 2 followed by functional assays can identify critical interaction residues. This approach has been successfully applied to study regulatory domains like the αCTD in mycobacterial ATP synthases .

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique can map the solvent accessibility of different regions of subunit b 2, providing insights into which domains interact with other subunits and which are exposed to solvent.

When designing these experiments, researchers should consider that interactions may be dynamic and state-dependent, requiring analysis under different physiological conditions.

How can researchers assess the functional integrity of recombinant Maricaulis maris ATP synthase subunit b 2 after purification?

Assessing the functional integrity of recombinant ATP synthase subunit b 2 after purification presents unique challenges since the subunit itself does not possess enzymatic activity. Researchers should employ these complementary methods:

• Circular dichroism (CD) spectroscopy: This technique provides information about secondary structure content, confirming that the recombinant protein is properly folded.

• Reconstitution assays: The ultimate test of functionality involves reconstituting the purified subunit b 2 with other ATP synthase components and measuring the ATP synthesis/hydrolysis activity of the reconstituted complex. Decreased activity compared to reconstitution with native subunit b 2 would indicate compromised functionality.

• Binding assays with interaction partners: Using techniques such as microscale thermophoresis (MST) or isothermal titration calorimetry (ITC) to quantify binding affinities between subunit b 2 and known interaction partners can provide evidence of functional integrity.

• Limited proteolysis: Properly folded proteins often display characteristic resistance patterns to proteolytic digestion. Changes in digestion patterns can indicate structural abnormalities.

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS): This can verify that the protein maintains its expected oligomeric state, which is critical for function within the ATP synthase complex.

Unlike analyses of catalytic subunits where direct activity measurements are possible , assessment of structural subunits like b 2 relies more heavily on structural integrity and protein-protein interaction analyses.

What experimental approaches can elucidate the role of ATP synthase subunit b 2 in energy metabolism of Maricaulis maris?

To investigate the specific role of ATP synthase subunit b 2 in Maricaulis maris energy metabolism, researchers should consider these experimental approaches:

• Gene knockout/knockdown studies: Create atpF2 deletion or conditional expression strains and analyze changes in growth rates, ATP production, and proton gradient maintenance under different metabolic conditions.

• Site-directed mutagenesis of conserved residues: Based on structural data or sequence alignments, mutate key residues and analyze effects on ATP synthesis efficiency, similar to studies conducted with mycobacterial ATP synthase components .

• In vivo cross-linking followed by immunoprecipitation: This can identify novel interaction partners of subunit b 2 under different metabolic conditions, providing insights into its regulatory roles beyond structural support.

• Metabolic flux analysis: Compare metabolic pathway utilization between wild-type and atpF2-mutant strains to understand how alterations in ATP synthase function affect global cellular metabolism.

• Membrane potential measurements: Using fluorescent probes, measure how mutations in subunit b 2 affect the ability of Maricaulis maris to maintain membrane potential under different energetic conditions.

• Structural studies of the intact ATP synthase complex: Using techniques like cryo-EM (as applied to mycobacterial ATP synthase ), compare the structure of wild-type complexes with those containing mutated subunit b 2 to understand structural consequences.

These approaches collectively would provide a comprehensive understanding of how subunit b 2 contributes to energy metabolism in Maricaulis maris.

What implications do ATP synthase inhibitors have for research on bacterial energy metabolism?

ATP synthase inhibitors have become powerful tools for studying bacterial energy metabolism and represent potential antimicrobial agents. Their research implications include:

• Selective targeting: Structural differences between bacterial ATP synthases, like the mycobacterium-specific γ-loop and αCTD , provide opportunities for species-specific inhibitors with minimal effects on human ATP synthase, reducing potential toxicity.

• Metabolic vulnerability identification: Inhibitor studies have revealed that bacteria like Mycobacterium tuberculosis are particularly vulnerable to ATP depletion when in dormant states, as demonstrated with inhibitors like bedaquiline (BDQ) and GaMF1 that target mycobacterial ATP synthase .

• Resistance mechanism elucidation: Research with ATP synthase inhibitors has uncovered novel resistance mechanisms, providing insights into bacterial adaptation and evolution.

• Structural biology advancements: The development of inhibitors has driven structural studies of bacterial ATP synthases, including cryo-EM structures of inhibitor-bound complexes , advancing our understanding of the molecular mechanisms of ATP synthesis.

• Potential for combination therapies: Understanding how ATP synthase inhibition affects other metabolic pathways has revealed potential synergistic drug combinations targeting energy metabolism.

Studying how inhibitors interact with different subunits, including structural components like subunit b 2, can provide fundamental insights into the coupling mechanism between proton translocation and ATP synthesis.

What are the recommended methods for expression and purification of recombinant Maricaulis maris ATP synthase subunit b 2?

For optimal expression and purification of recombinant Maricaulis maris ATP synthase subunit b 2, researchers should consider the following methodological approach:

• Expression system selection:

  • E. coli BL21(DE3) or C43(DE3) strains are recommended for membrane protein expression

  • Consider using a fusion tag (His₆, MBP, or SUMO) to improve solubility and facilitate purification

  • The expression vector should contain a T7 promoter with tight regulation to control expression levels

• Culture conditions optimization:

  • Perform expression at lower temperatures (16-25°C) to improve proper folding

  • Induce with lower IPTG concentrations (0.1-0.5 mM) to prevent inclusion body formation

  • Consider autoinduction media for gentler protein expression

• Cell lysis and membrane preparation:

  • Use gentle lysis methods (French press or sonication with cooling intervals)

  • Separate membrane fraction by ultracentrifugation (100,000 × g for 1 hour)

  • Extract membrane proteins using mild detergents (DDM, LMNG, or C₁₂E₈)

• Purification strategy:

  • Initial capture: Immobilized metal affinity chromatography (IMAC)

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography

  • Consider incorporating a tag cleavage step if the tag might interfere with functional studies

• Quality control:

  • Assess purity by SDS-PAGE and Western blotting

  • Verify identity by mass spectrometry

  • Analyze secondary structure by circular dichroism spectroscopy

This methodology provides a starting point that should be optimized based on specific research requirements and the intended applications of the purified protein.

What techniques are most effective for studying the conformational dynamics of ATP synthase subunit b 2 during the catalytic cycle?

Understanding the conformational dynamics of ATP synthase subunit b 2 during the catalytic cycle requires sophisticated biophysical techniques. Based on approaches successfully applied to other ATP synthase components, the following methods are recommended:

• Single-molecule FRET (smFRET):

  • Label strategic positions in subunit b 2 with appropriate FRET pairs

  • Monitor distance changes during ATP synthesis/hydrolysis in real-time

  • This approach has been valuable for studying rotational dynamics in F₁-ATPase complexes from mycobacteria

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

  • Compare the exchange patterns under different catalytic states

  • Identify regions with altered solvent accessibility during the catalytic cycle

  • This provides detailed structural information without requiring protein modification

• Time-resolved cryo-EM:

  • Capture the ATP synthase complex in different catalytic states by rapid freezing

  • Analyze the conformational changes in subunit b 2 across these states

  • Recent advances have made this approach feasible for studying transient intermediates

• Site-directed spin labeling with EPR spectroscopy:

  • Introduce spin labels at strategic positions in subunit b 2

  • Measure changes in mobility and distance between labels during catalysis

  • Particularly useful for studying membrane-embedded portions of the protein

• Molecular dynamics simulations:

  • Use atomistic simulations to predict conformational changes in response to rotation

  • Validate computational predictions with experimental measurements

  • This approach can provide insights difficult to obtain experimentally

These techniques can be complementary, with computational approaches guiding experimental design and experimental results refining computational models.

How can researchers investigate potential species-specific variations in ATP synthase subunit b 2 structure and function?

Investigating species-specific variations in ATP synthase subunit b 2 requires a comparative approach combining bioinformatics, structural biology, and functional analysis:

• Comparative sequence analysis:

  • Perform multiple sequence alignments of subunit b 2 across diverse bacterial species

  • Identify conserved domains and species-specific insertions/deletions

  • Use evolutionary trace methods to identify functionally important residues

  • Create phylogenetic trees to understand evolutionary relationships

• Structural comparison:

  • Generate homology models based on existing ATP synthase structures

  • Compare these models to identify structural differences

  • If possible, determine structures of subunit b 2 from multiple species using cryo-EM or X-ray crystallography

  • Special attention should be paid to regions analogous to the regulatory domains identified in mycobacterial ATP synthases

• Domain swapping experiments:

  • Create chimeric proteins with domains from different species

  • Test functionality in reconstituted systems

  • This approach has been used successfully to study the function of the mycobacterium-specific γ-loop

• Heterologous expression studies:

  • Express Maricaulis maris subunit b 2 in different bacterial hosts

  • Compare functionality to identify host-specific compatibility factors

  • Assess how species-specific factors affect assembly and function

• Functional comparison across species:

  • Compare ATP synthesis rates, proton conductance, and inhibitor sensitivity

  • Correlate functional differences with structural variations

  • Identify adaptations related to specific environmental niches

This multi-faceted approach would provide insights into how evolutionary pressures have shaped ATP synthase structure and function across bacterial species.

How can structural information about ATP synthase subunit b 2 contribute to the development of new antimicrobial agents?

The structural features of ATP synthase subunit b 2 present unique opportunities for antimicrobial drug development, particularly for targeting bacteria with species-specific characteristics:

• Targeting protein-protein interfaces:

  • The interface between subunit b 2 and other components of the ATP synthase complex represents a potential site for small molecule inhibitors

  • Unlike active site inhibitors, interface inhibitors may achieve greater species specificity

  • Similar approaches targeting mycobacterial-specific interfaces have shown promise

• Exploiting species-specific structural elements:

  • Just as the mycobacterial γ-loop and αCTD provide targets for species-specific inhibitors like GaMF1 , unique structural features of subunit b 2 in pathogenic bacteria could be exploited

  • Comparative structural analysis can identify such unique features

• Disrupting assembly rather than function:

  • Small molecules that interfere with the incorporation of subunit b 2 into the ATP synthase complex could prevent proper assembly

  • This approach potentially offers greater specificity than inhibiting the conserved catalytic mechanism

• Structure-based design considerations:

  • High-resolution structural data is essential for rational design of inhibitors

  • Molecular dynamics simulations can identify transiently exposed binding pockets

  • Fragment-based drug discovery approaches are particularly suited for targeting protein-protein interfaces

• Combination approaches:

  • Inhibitors targeting different components of ATP synthase might synergize

  • The success of bedaquiline against mycobacterial ATP synthase demonstrates the clinical potential of ATP synthase inhibitors

The development of such inhibitors could provide new therapeutic options against multidrug-resistant bacteria, particularly those where existing ATP synthase inhibitors like bedaquiline show limited efficacy.

What role might post-translational modifications play in regulating ATP synthase subunit b 2 function?

Post-translational modifications (PTMs) represent an understudied aspect of ATP synthase regulation that may have significant implications for understanding bacterial energy metabolism:

• Potential PTMs affecting subunit b 2:

  • Phosphorylation: Could alter structural rigidity or interactions with other subunits

  • Acetylation: Might affect the charge distribution and interaction properties

  • Oxidative modifications: As seen in mitochondrial ATP synthase α subunit in Alzheimer's disease , oxidative damage could affect function

  • Lipid modifications: Could influence membrane association

• Methodological approaches for PTM identification:

  • Mass spectrometry-based proteomics with enrichment for specific modifications

  • Site-directed mutagenesis of potential modification sites to mimic or prevent modifications

  • In vitro modification assays to assess functional consequences

• Regulatory implications:

  • PTMs could provide a rapid response mechanism to changing environmental conditions

  • Different from the structural inhibitory mechanisms seen with the αCTD in mycobacteria , PTMs might offer more dynamic and reversible regulation

  • PTMs might coordinate ATP synthase activity with other metabolic processes

• Species-specific considerations:

  • The pattern of PTMs might differ between species, contributing to adaptation to different environments

  • The enzymes responsible for these modifications could be species-specific, offering potential drug targets

• Technical challenges:

  • Low abundance of modifications may require sensitive detection methods

  • Preserving labile modifications during protein purification requires careful protocol optimization

This represents a frontier area in ATP synthase research with potential implications for understanding bacterial adaptation and energy regulation.

How should researchers interpret and troubleshoot unexpected results when working with recombinant ATP synthase components?

When working with recombinant ATP synthase components like subunit b 2, researchers frequently encounter unexpected results. Here's a systematic approach to interpretation and troubleshooting:

When unexpected results occur with ATP synthase subunit b 2, it's particularly important to consider:

• The membrane-associated nature of the protein may require specialized handling

• Interactions with other subunits may be necessary for stability and proper folding

• The native lipid environment may be critical for maintaining physiological conformation

Researchers should systematically document all experimental conditions and variations, as seemingly minor changes can significantly impact results with complex multi-subunit systems like ATP synthase.

What are the main challenges in correlating in vitro findings about ATP synthase subunit b 2 with its in vivo function?

Translating in vitro observations about ATP synthase subunit b 2 to in vivo function presents several significant challenges:

• Reconstitution limitations:

  • In vitro systems often lack the complete cellular context

  • The lipid composition used for reconstitution may not match the native membrane environment

  • The precise stoichiometry of components may differ from natural systems

  • Similar challenges have been noted in studies of mycobacterial ATP synthase components

• Regulatory network absence:

  • In vivo, ATP synthase activity is regulated by numerous factors including metabolic state, ion gradients, and regulatory proteins

  • These complex regulatory networks are difficult to recreate in vitro

• Technical considerations:

  • The energetic parameters (ΔpH, Δψ) in vitro may not match physiological conditions

  • Mutations that appear to affect function in vitro may be compensated for in vivo

  • The time scales of in vitro experiments often differ from physiological processes

• Validation approaches:

  • Complement in vitro studies with genetic approaches (gene deletion, complementation)

  • Use site-directed mutagenesis to test specific hypotheses in both systems

  • Develop intermediate complexity systems (spheroplasts, inverted membrane vesicles)

  • Apply in vivo imaging or labeling techniques to track ATP synthase assembly and localization

• Interpretation guidelines:

  • Establish clear correlations between in vitro parameters and in vivo phenotypes

  • Consider multiple in vivo readouts (growth rates, ATP levels, membrane potential)

  • Be cautious about extrapolating from single subunit studies to whole complex function

Addressing these challenges requires integrating multiple experimental approaches and developing more sophisticated in vitro systems that better mimic the cellular environment.