Recombinant Bacillus PS3 ATP synthase subunit b (atpF)

What is the structural position of subunit b in Bacillus PS3 ATP synthase?

Structural studies of Bacillus PS3 ATP synthase have revealed that in the F₀ region, one copy of subunit b is positioned at a location equivalent to that of the mitochondrial subunit b, while the second copy occupies the position analogous to yeast subunit 8 (mammalian A6L) on the opposite side of subunit a . This structural arrangement is fundamental to understanding the architecture of bacterial ATP synthases compared to their mitochondrial counterparts.

How many copies of subunit b are present in the Bacillus PS3 ATP synthase complex?

The Bacillus PS3 ATP synthase complex contains two copies of subunit b (b₂), unlike some other F-type ATP synthases that may have different stoichiometry . These two copies form part of the peripheral stalk of the ATP synthase, helping to connect the F₁ and F₀ regions of the complex.

What expression system has been successfully used for recombinant Bacillus PS3 ATP synthase?

Researchers have successfully expressed Bacillus PS3 ATP synthase, including subunit b with an N-terminal 10×His tag, using a plasmid expression system in Escherichia coli strain DK8 . This E. coli strain is particularly suitable as it lacks endogenous ATP synthase (due to deletion of the unc operon), eliminating potential contamination from the host ATP synthase during purification .

What is the recommended purification protocol for recombinant Bacillus PS3 ATP synthase containing subunit b?

Based on published methodologies, the following purification protocol has proven effective:

• Express Bacillus PS3 ATP synthase in E. coli strain DK8

• Extract the complex from membranes using a suitable detergent

• Purify using metal-affinity chromatography, exploiting the N-terminal 10×His tag on subunit b

• Further purify the complex as needed for downstream applications (e.g., structural studies)

This approach yields intact ATP synthase complexes suitable for structural and functional studies, including cryo-EM analysis.

How can researchers visualize the structure of Bacillus PS3 ATP synthase and its subunit b?

High-resolution structural visualization of Bacillus PS3 ATP synthase, including subunit b, has been achieved using cryo-electron microscopy (cryo-EM) . The protocol involves:

• Preparation of purified ATP synthase complexes

• Cryo-EM grid preparation and data collection

• 3D classification to identify different rotational states

• Refinement to high resolution (approximately 3.0 Å)

For focused studies on the F₀ region containing subunit b, additional focused refinement of the membrane-embedded region can improve resolution for this portion of the complex .

What is the structural relationship between the two copies of subunit b in Bacillus PS3 ATP synthase?

Contrary to earlier hypotheses based on cross-linking experiments that suggested proximity between the N-termini of the two b subunits, high-resolution structural data reveals that the transmembrane α-helices of the two b-subunits are actually positioned on opposite sides of subunit a . This finding suggests that previous cross-linking results may have captured non-specific interactions between b-subunits from neighboring ATP synthase complexes rather than interactions within a single complex .

How do the two copies of subunit b interact with subunit a?

The single N-terminal membrane-embedded α-helix in each of the two copies of subunit b forms distinct interactions with subunit a:

These differential interactions explain why mutations in this region often impact both assembly and activity of the complex .

What role does subunit b play in the flexibility of the ATP synthase complex?

Analysis of the three rotational states of Bacillus PS3 ATP synthase reveals that the C-terminal water-soluble part of subunit b displays significant conformational variability between states . This suggests that subunit b contributes to the flexibility of the peripheral stalk, which may be important for accommodating the conformational changes that occur during rotary catalysis.

Specifically, comparison of the three rotational states shows that:

• The C-terminal water-soluble part of subunit b shows the most significant conformational variability

• The peripheral stalk in Bacillus PS3 is structurally simpler and more flexible than in yeast mitochondria

• This flexibility may be important for the function of the enzyme during rotation

How does the structural simplicity of bacterial subunit b compare to mitochondrial equivalents?

The peripheral stalk containing subunit b in Bacillus PS3 ATP synthase is structurally simpler and more flexible than its counterpart in yeast mitochondria . This observation suggests that in bacterial ATP synthases, the c-ring and subunit a are primarily held together by hydrophobic interactions rather than by the peripheral stalk, which differs from the more complex arrangements in mitochondrial systems .

How can site-directed mutagenesis of subunit b inform our understanding of ATP synthase assembly and function?

Given the distinct interactions formed by the two copies of subunit b with subunit a, site-directed mutagenesis studies targeting specific residues at these interfaces could provide valuable insights into:

When designing such experiments, researchers should consider:

What are the implications of subunit b flexibility for the mechanism of ATP synthesis/hydrolysis?

The observed flexibility in the C-terminal water-soluble part of subunit b may have important implications for understanding the mechanism of ATP synthesis and hydrolysis:

• The flexibility may allow the peripheral stalk to accommodate the rotational movements of the central stalk during catalysis

• It may contribute to the transient storage of torsional energy during rotation

• It may influence the coupling between proton translocation in F₀ and ATP synthesis/hydrolysis in F₁

Future research could explore how modulating this flexibility affects enzyme kinetics and efficiency .

How do the three rotational states observed in cryo-EM structures relate to the catalytic cycle?

The three rotational states of Bacillus PS3 ATP synthase captured by cryo-EM reveal distinct angular positions of the rotor (subunits γεc₁₀) . The structure shows a symmetry mismatch between the 120° steps of the F₁ motor and 36° steps of the F₀ motor, with the rotational step sizes in the three states appearing to be almost exactly 3, 4, and 3 c-subunits .

This rotational mechanism has implications for understanding:

• The coupling between proton translocation and ATP synthesis

• The role of subunit b in stabilizing the complex during rotation

• The mechanical transmission of energy between F₀ and F₁ regions

How can the structural insights of Bacillus PS3 ATP synthase subunit b inform drug discovery targeting bacterial ATP synthases?

The structural differences between bacterial ATP synthases (including the arrangement and interactions of subunit b) and their mitochondrial counterparts provide potential targets for selective inhibition. Researchers could:

• Target the unique interfaces between subunit b and subunit a

• Exploit the differential flexibility of the bacterial peripheral stalk

• Design compounds that interfere with the assembly of the bacterial complex without affecting mitochondrial ATP synthase

Such approaches could lead to novel antibiotics that selectively target bacterial energy metabolism .

What experimental approaches can be used to study the dynamics of subunit b in functional ATP synthase?

Several experimental approaches can provide insights into the dynamics of subunit b during ATP synthesis/hydrolysis:

• Single-molecule FRET: By labeling specific residues in subunit b, researchers can monitor conformational changes during catalysis

• Cross-linking studies: Strategic cross-linking of subunit b to other subunits can capture transient interactions during the catalytic cycle

• Molecular dynamics simulations: Based on the high-resolution structures, simulations can predict dynamic behaviors not captured by static structures

• Hydrogen-deuterium exchange mass spectrometry: This technique can identify regions of flexibility and solvent exposure in the functioning complex

These approaches would complement the static structural information provided by cryo-EM studies.

What factors affect the expression yield of recombinant Bacillus PS3 ATP synthase with tagged subunit b?

Several factors can influence the expression yield of recombinant Bacillus PS3 ATP synthase:

• E. coli strain selection: DK8 strain is preferred as it lacks endogenous ATP synthase

• Expression temperature: Lower temperatures (25-30°C) may improve proper folding and assembly

• Induction conditions: IPTG concentration and induction timing should be optimized

• Media composition: Rich media may improve yields but could affect specific activity

• Position and nature of the affinity tag: The 10×His tag on the N-terminus of subunit b has been successfully used, but other positions or tag types might affect assembly

How can researchers distinguish between properly assembled and misassembled ATP synthase complexes containing subunit b?

Distinguishing between properly assembled and misassembled complexes is crucial for structural and functional studies: