Recombinant Lysinibacillus sphaericus ATP synthase subunit b (atpF)

What is the function of ATP synthase subunit b in Lysinibacillus sphaericus?

ATP synthase subunit b in L. sphaericus serves as a critical component of the F₀F₁ ATP synthase complex, which is responsible for ATP production during oxidative phosphorylation. Specifically, the b subunit forms part of the peripheral stalk that connects the F₁ catalytic domain to the F₀ membrane domain . The b subunit exists as a dimer in the functional complex and helps prevent rotation of the F₁ domain, enabling the enzyme to convert the proton gradient energy into ATP synthesis .

The structure and function of this subunit must be considered within the context of L. sphaericus' unique metabolism, which relies on acetate and glycerol rather than carbohydrates as carbon sources . This specialized metabolism affects the organism's energy production pathways, making ATP synthase particularly important for its survival.

What structural characteristics define the ATP synthase subunit b from L. sphaericus?

Based on analytical studies, the ATP synthase subunit b dimerization domain (residues 62-122) exhibits distinct structural features:

These structural characteristics are consistent with an alpha-helical coiled-coil architecture , which provides the mechanical stability necessary for the b subunit's role in the ATP synthase complex. Interestingly, while the functional form is dimeric, crystallographic studies show a monomeric form, suggesting potential conformational changes during complex assembly .

Why would researchers be interested in studying L. sphaericus atpF specifically?

Researchers might focus on L. sphaericus atpF for several compelling reasons:

• Unique metabolic context: L. sphaericus cannot metabolize carbohydrates due to the absence of a key enzyme (EC 5.3.1.9) , making its energy production pathways particularly interesting.

• Biotechnological applications: L. sphaericus has significant potential for mosquito biocontrol and bioremediation of toxic metals , and understanding its energy metabolism could enhance these applications.

• Evolutionary insights: Studying atpF in this organism could provide insights into how ATP synthase adapts to specialized metabolic requirements.

• Structural biology: The dimerization properties of the b subunit present an interesting model for studying protein-protein interactions in membrane protein complexes .

• Comparative biochemistry: Differences between L. sphaericus ATP synthase and better-studied homologs could reveal alternative mechanisms for this essential molecular machine.

How does the dimerization of ATP synthase subunit b affect its function in L. sphaericus?

The dimerization of ATP synthase subunit b is fundamental to its function within the ATP synthase complex. In L. sphaericus, as in other organisms, this dimerization specifically involves residues 62-122 , creating an elongated structure with distinct mechanical properties.

Functionally, this dimerization:

• Provides mechanical stability to the peripheral stalk

• Prevents rotation of the F₁ domain during catalysis

• Maintains proper alignment between F₁ and F₀ components

• May participate in energy transfer within the complex

Research approaches to study this dimerization include:

• Site-directed mutagenesis of key residues within the 62-122 region

• Cross-linking studies followed by mass spectrometry to identify interaction surfaces

• Comparative analysis with b subunits from other species to identify conserved dimerization motifs

• Single-molecule force spectroscopy to measure the strength of dimer interactions

Understanding how the unique metabolic adaptations of L. sphaericus might influence the structure and function of this dimer represents an important research direction, particularly given the organism's inability to utilize conventional carbon sources .

What expression systems are most suitable for producing recombinant L. sphaericus atpF for structural studies?

Selecting an appropriate expression system for L. sphaericus atpF requires careful consideration of multiple factors, especially when the goal is structural characterization. The following approaches offer distinct advantages:

For crystallographic studies similar to those performed on the dimerization domain (residues 62-122) , expression of this specific fragment rather than the full-length protein often yields better results. E. coli expression with a cleavable tag (His, MBP, or SUMO) followed by tag removal through specific proteases typically provides the best material for crystallization trials.

For studying interactions within the ATP synthase complex, co-expression of multiple subunits might be necessary to obtain stable, properly folded protein. This approach would better reflect the native context where the b subunit exists as a dimer within a larger macromolecular assembly .

How might the iron metabolism of L. sphaericus affect experiments with recombinant atpF?

L. sphaericus possesses a sophisticated iron acquisition and metabolism system, including siderophore production, iron transporters, and regulatory mechanisms . This specialized iron metabolism could significantly impact recombinant expression of atpF and subsequent experimental approaches in several ways:

Researchers should monitor iron levels and consider how the nine identified Fur-binding boxes in L. sphaericus might regulate expression of atpF or other components of energy metabolism pathways under different experimental conditions.

What purification strategies are most effective for isolating recombinant L. sphaericus atpF?

Purifying recombinant L. sphaericus ATP synthase subunit b requires a strategic approach that accounts for its structural characteristics and tendency to dimerize. An effective purification strategy might include:

Step 1: Initial extraction

• For membrane-associated full-length atpF: Detergent screening (DDM, LDAO, or C12E8)

• For soluble constructs (e.g., dimerization domain residues 62-122): Standard lysis buffers

Step 2: Primary purification

• Affinity chromatography: His-tag purification (IMAC) with imidazole gradient elution

• Optimization for the elongated structure: Reduced flow rates and extended binding times

Step 3: Intermediate purification

• Ion exchange chromatography: Based on the predicted isoelectric point

• Tag removal: Precision protease treatment followed by reverse IMAC

Step 4: Polishing

• Size exclusion chromatography: Critical for separating monomeric and dimeric forms

• Buffer optimization: Screening conditions that maintain the native oligomeric state

Special considerations:

• The extremely elongated structure (frictional ratio of 1.60) may cause anomalous migration on size exclusion columns

• The dimerization domain should be handled carefully to preserve its alpha-helical coiled-coil structure

• Analytical ultracentrifugation may be necessary to confirm proper assembly of the purified protein

For crystallography purposes similar to the previous 1.55 Å structure , additional steps focused on homogeneity and concentration would be required, potentially including crystallization chaperones or fusion partners to promote crystal contacts.

What techniques are available for studying the interaction between atpF and other ATP synthase components?

Understanding the interactions between atpF and other ATP synthase components requires a multi-methodological approach:

For studying the dimerization domain specifically:

Researchers should note that the monomeric state observed in crystal structures versus the dimeric functional form highlights the importance of validating interactions using multiple complementary techniques.

How can researchers investigate the role of atpF in the context of L. sphaericus' unique metabolism?

L. sphaericus has a distinctive metabolism characterized by an inability to metabolize carbohydrates and reliance on alternative carbon sources like acetate and glycerol . Investigating atpF's role within this metabolic context requires specialized approaches:

Genetic approaches:

• Gene knockout/knockdown of atpF followed by metabolic profiling

• Site-directed mutagenesis of key residues to create partially functional variants

• Complementation studies with atpF homologs from organisms with different metabolic capabilities

Biochemical approaches:

• ATP synthesis/hydrolysis assays using membrane vesicles or reconstituted systems

• Respiration measurements with various carbon sources (acetate, glycerol)

• Proton pumping assays to assess coupling efficiency

Systems biology approaches:

• Integration with metabolic models of L. sphaericus

• Transcriptomic analysis under different growth conditions

• Proteomic analysis of ATP synthase complex composition and abundance

Specific experimental considerations:

• Compare ATP synthesis efficiency with acetate versus other potential carbon sources

• Assess how nitrogen metabolism (e.g., from ethanolamine, threonine, and glycine) affects ATP synthase function

• Investigate potential relationships between iron metabolism and oxidative phosphorylation efficiency

Since L. sphaericus cannot utilize conventional carbohydrates , researchers should design experiments that account for this metabolic constraint when studying energy conversion through ATP synthase.

How should researchers interpret structural differences between monomeric crystal forms and functional dimers of L. sphaericus atpF?

The crystal structure of the ATP synthase b subunit dimerization domain (residues 62-122) shows an isolated, monomeric alpha helix of 90 Å length , while functional and solution studies indicate a dimeric coiled-coil structure. This apparent discrepancy requires careful interpretation:

Possible explanations for the structural differences:

• Crystal packing forces may disrupt the natural dimerization

• Crystallization conditions might favor monomeric states

• The construct used for crystallization may lack elements that stabilize the dimer

• The observed monomer may represent an assembly intermediate

Approaches for reconciling these differences:

• Solution studies: Analytical ultracentrifugation and SAXS data showing elongated dimers (frictional ratio 1.60, maximal dimension 95 Å) provide context for interpreting crystal structures

• Molecular dynamics simulations: Can help model the transition between monomeric and dimeric states

• Alternative crystallization strategies: Co-crystallization with interacting partners or cross-linking prior to crystallization

• Integrative structural biology: Combining multiple techniques (X-ray, cryo-EM, SAXS, NMR) to build a comprehensive structural model

Researchers should consider these structural studies within the context of L. sphaericus' unique biology, including its specialized metabolism and how this might influence ATP synthase structure and function compared to better-characterized systems.

What factors should be considered when comparing L. sphaericus atpF to homologs from other bacterial species?

When comparing L. sphaericus atpF to homologs from other bacterial species, researchers should consider several key factors:

Genomic and evolutionary context:

• L. sphaericus has been proposed as a novel species based on comparative genomic analysis

• The specialized metabolism of L. sphaericus, particularly its inability to utilize carbohydrates , may drive unique adaptations in energy production systems

Structural considerations:

• The dimerization domain (residues 62-122) forms an elongated structure that may differ in length or stability from homologs

• Alpha-helical coiled-coil motifs may show species-specific variations in helix packing or surface residues

Functional aspects:

• ATP synthase efficiency may be optimized for L. sphaericus' reliance on acetate and glycerol metabolism

• Interactions with other ATP synthase components might show adaptations specific to L. sphaericus

Methodological approach for comparison:

• Sequence alignment focusing on the dimerization domain (residues 62-122)

• Structural superposition of available crystal structures

• Phylogenetic analysis in the context of metabolic capabilities

• Functional complementation experiments

The table below summarizes potential key differences to examine:

This comparative approach can provide insights into how ATP synthase has adapted to the specialized ecological niche and metabolic constraints of L. sphaericus.

How can researchers investigate the potential relationship between L. sphaericus' iron metabolism and ATP synthase function?

L. sphaericus possesses an extensive iron acquisition and metabolism system , which might interact with or influence ATP synthase function. Investigating this relationship requires specialized approaches:

Experimental strategies:

• Growth and expression under iron-limited versus iron-replete conditions

• Mutation of Fur-binding sites identified in L. sphaericus followed by ATP synthase activity assays

• Metabolomic profiling to identify links between iron status and energy metabolism

• Proteomic analysis of ATP synthase complex composition under varying iron conditions

Specific techniques:

• ICP-MS to quantify iron associated with purified ATP synthase complex

• EPR spectroscopy to detect potential iron-sulfur clusters in proximity to ATP synthase

• Transcriptomic analysis focusing on atpF expression in response to iron availability

• ATP synthesis assays in the presence of iron chelators or siderophores produced by L. sphaericus

The nine identified Fur-binding boxes in L. sphaericus genomes should be examined for proximity to atpF or other ATP synthase components, as these might indicate direct regulation by iron availability.

This research direction could reveal novel regulatory mechanisms linking environmental iron availability to energy production in L. sphaericus.