Recombinant Bacillus PS3 ATP synthase protein I (atpI)

What is the basic structure of Bacillus PS3 ATP synthase and how does it differ from other bacterial ATP synthases?

Bacillus PS3 ATP synthase (TF0F1) is a multi-subunit enzyme complex that produces ATP from ADP and inorganic phosphate using energy from a transmembrane proton motive force. It consists of two main portions: the membrane-embedded F0 portion that conducts protons and the soluble F1 portion that catalyzes ATP synthesis .

Recent cryo-EM studies have revealed atomic models of the complex in three rotational states with resolutions of 3.0-3.2 Å, providing insights into how this seemingly simple bacterial ATP synthase performs the same core functions as more complex mitochondrial versions . The architecture shows how loops in subunit a of the bacterial enzyme functionally replace additional subunits found in mitochondrial enzymes.

Compared to other bacterial ATP synthases, Bacillus PS3 exhibits distinctive characteristics:

What expression systems are most effective for producing recombinant Bacillus PS3 ATP synthase?

Recombinant Bacillus PS3 ATP synthase has been successfully expressed in E. coli expression systems, allowing for genetic manipulation and biochemical analysis . The thermostability of the enzyme makes it particularly amenable to expression in mesophilic hosts.

For optimal expression:

• The complete ATP synthase operon is typically cloned into appropriate expression vectors

• Modifications such as an N-terminal 10× His tag on subunit β facilitate purification

• Expression is performed under controlled temperature and induction conditions

• The thermostability of Bacillus PS3 proteins allows for heat treatment during purification to eliminate E. coli contaminants

E. coli expression systems have consistently yielded functional enzyme suitable for structural studies (including cryo-EM analysis) and reconstitution into artificial systems .

What methodological approaches are recommended for purifying functional Bacillus PS3 ATP synthase?

Purification of recombinant Bacillus PS3 ATP synthase requires careful handling to maintain structural integrity and enzymatic activity. The following methodology has proven effective:

• Cell disruption: Mechanical disruption of E. coli cells expressing the recombinant protein

• Membrane preparation: Isolation of bacterial membranes containing the ATP synthase complex

• Detergent solubilization: The choice of detergent is critical - octyl glucoside and Triton X-100 have shown superior results for extracting functional protein

• Affinity chromatography: Utilizing the His-tag on subunit β for selective purification

• Additional chromatography: Size exclusion or ion exchange chromatography for improved purity

• Quality assessment: SDS-PAGE analysis and enzyme activity assays

The purified enzyme can be maintained in a stabilizing buffer containing appropriate detergents, or immediately reconstituted into liposomes for functional studies .

How can researchers verify the proper folding and assembly of recombinant Bacillus PS3 ATP synthase?

Verification of proper folding and assembly requires multiple analytical approaches:

• Enzymatic activity assays: Measuring ATP synthesis/hydrolysis rates under controlled conditions. Properly assembled enzymes can achieve activities of 500-800 nmol ATP × min⁻¹ × mg TF0F1⁻¹ in optimized reconstituted systems

• Structural analysis:

  • Negative stain electron microscopy for initial quality assessment

  • Cryo-EM for high-resolution structural verification

  • The characteristic "lollipop" shape of F1 attached to the membrane-embedded F0 is visible in properly assembled complexes

• Subunit composition verification:

  • SDS-PAGE analysis should reveal all expected subunits in appropriate stoichiometry

  • Western blotting with subunit-specific antibodies

• Proton pumping assays: When reconstituted into liposomes, functional ATP synthase should demonstrate proton translocation coupled to ATP synthesis/hydrolysis

What are the optimal conditions for reconstitution of Bacillus PS3 ATP synthase into functional proteoliposomes?

Reconstitution of Bacillus PS3 ATP synthase into proteoliposomes requires careful optimization of multiple parameters:

• Liposome composition: Phosphatidylcholine/phosphatidic acid liposomes prepared by reverse-phase evaporation provide an effective membrane system. Negatively charged phospholipids are essential for light-driven ATP synthesis

• Detergent selection and protein insertion protocol:

  • Initial studies tested various detergents: Triton X-100, octyl glucoside, octaethylene glycol n-dodecylether, sodium cholate, and sodium deoxycholate

  • The most efficient reconstitutions were achieved with octyl glucoside or Triton X-100

  • An optimized approach involves preparing empty liposomes first, then adding purified protein before complete detergent removal

  • This method achieves approximately 70% proper orientation of membrane proteins

• Activation protocol: A critical step involves activation of the highly stable TF0F1 through total solubilization of phospholipids and proteins in a Triton X-100/octyl glucoside mixture containing 20 mM octyl glucoside, leading to a threefold stimulation of ATP synthase activity

• Membrane composition enhancements: Adding cholesterol induces a fourfold increase in ATP synthase activity with a concurrent 65% decrease in the Km for ADP (from 330 μM to 115 μM)

Following these optimized protocols, researchers have achieved ATP synthase activities up to 20-fold higher than previously reported values for light-driven systems .

How can cryo-EM be effectively used to resolve the structure of Bacillus PS3 ATP synthase in different conformational states?

Cryo-electron microscopy has revolutionized structural studies of Bacillus PS3 ATP synthase, revealing atomic details of the complex in different rotational states:

• Sample preparation optimization:

  • Protein concentration: 0.5-3 mg/ml in detergent micelles

  • Grid preparation: Application of 3-4 μl sample to glow-discharged grids

  • Vitrification: Rapid freezing in liquid ethane using automated plunging devices

• Data collection parameters:

  • Voltage: 300 kV electron microscopes

  • Magnification: 22,500-29,000×

  • Pixel size: 1.05-1.08 Å

  • Dose: 40-60 e-/Å2 total exposure

  • Frame collection: 40 frames per exposure for motion correction

• Image processing workflow:

  • Motion correction and CTF estimation

  • Particle picking (250,000-300,000 particles)

  • 2D and 3D classification to separate conformational states

  • Refinement to achieve 3.0-3.2 Å resolution

  • Model building and refinement

This approach has revealed critical structural insights, including the conformation of subunit ε in its inhibitory state, the architecture of the proton channel, and the detailed interactions between subunits . The three different rotational states captured represent snapshots of the enzyme during its catalytic cycle.

What molecular mechanisms underlie the regulatory function of the ε subunit in Bacillus PS3 ATP synthase?

The ε subunit serves as a critical regulator of Bacillus PS3 ATP synthase activity through ATP-dependent conformational changes:

• Conformational states:

  • "Up" conformation: C-terminal domain extends upward and inserts into the αDPβDP interface, forcing β to adopt an open conformation that inhibits ATP hydrolysis

  • "Down" conformation: C-terminal domain folds back against the N-terminal domain, allowing normal catalytic cycling

• ATP concentration dependence:

  • Low ATP (<0.7 mM): Promotes inhibitory "up" conformation

  • High ATP (>1 mM): Induces permissive "down" conformation

• Structural features revealed by cryo-EM:

  • The C-terminal part of subunit ε is entirely α-helical in Bacillus PS3

  • This differs from earlier crystal structure models that showed two α-helical segments broken at Ser 106

• Functional significance:

  • Prevents wasteful ATP hydrolysis during energy-limited conditions

  • Allows reverse operation (ATP hydrolysis to generate proton motive force) only when ATP is abundant

  • This mechanism differs from that in E. coli, where inhibition persists regardless of ATP concentration when proton motive force is insufficient

This regulatory mechanism represents a sophisticated cellular adaptation to balance energy production and consumption according to metabolic demands.

How can Bacillus PS3 ATP synthase be integrated into artificial photosynthetic systems for energy production?

Integration of Bacillus PS3 ATP synthase into artificial photosynthetic systems involves several sophisticated steps:

• Component preparation and purification:

  • FoF1 ATP synthase from Bacillus PS3 expressed in E. coli

  • Bacteriorhodopsin (bR) isolated from Halobacterium salinarum purple membrane

  • Phosphatidylcholine lipids (typically from soybean extract)

• Optimized proteoliposome formation:

  • Empty liposomes roughly preformed

  • Purified proteins added before complete detergent removal

  • This method achieves ~70% proper orientation of bacteriorhodopsin versus only 25% with conventional methods

  • Proper orientation directly correlates with improved proton-pump activity

• Construction of the artificial photosynthetic cell:

  • Small proteoliposomes containing ATP synthase and bacteriorhodopsin encapsulated within giant unilamellar vesicles

  • Cell-free protein synthesis system (PURE system) incorporated into the vesicle

  • This creates a system where light energy drives ATP production, which powers protein synthesis

• Self-sustaining system design:

  • Light activation of bacteriorhodopsin creates a proton gradient

  • ATP synthase uses this gradient to produce ATP

  • The synthesized ATP powers transcription and translation

  • The system can synthesize its own components, including bacteriorhodopsin or constituent proteins of ATP synthase

  • Newly synthesized proteins integrate into artificial organelles, enhancing photosynthetic activity through positive feedback

This approach has successfully demonstrated energy-independent protein synthesis within an artificial cell-like system, representing a significant advancement in synthetic biology .

How do specific amino acid residues in Bacillus PS3 ATP synthase affect ATP binding and regulatory function?

Mutational analysis has identified several critical residues in Bacillus PS3 ATP synthase that influence ATP binding and regulation:

• Key residues in the ε subunit ATP binding motif:

• Comparative analysis across species:

  • Despite high sequence similarity in the ATP binding motif between Bacillus PS3 and B. subtilis, they exhibit a 500-fold difference in ATP binding affinity (4 mM versus 2 mM)

  • E. coli ε subunit harbors four divergences from Bacillus PS3: E83I, R99K, R122K, and R126Q

  • These subtle sequence variations have profound effects on regulatory behavior

• Structural implications:

  • An allosteric Mg2+ binding site influences ATP binding affinity

  • The positioning of key arginine residues creates a positively charged pocket for ATP binding

  • The specific orientation of these residues determines the affinity and regulatory response to ATP

These structure-function relationships provide valuable insights for engineering ATP synthases with modified regulatory properties for biotechnological applications.

What strategies can address low activity or improper assembly of recombinant Bacillus PS3 ATP synthase?

Researchers encountering issues with recombinant Bacillus PS3 ATP synthase activity or assembly should consider these methodological approaches:

• Expression optimization:

  • Adjust induction conditions (temperature, IPTG concentration, duration)

  • Consider using specialized E. coli strains with enhanced protein folding capabilities

  • Co-express molecular chaperones to assist proper folding

• Purification troubleshooting:

  • Maintain strict temperature control during membrane preparation

  • Test different detergent types and concentrations for solubilization

  • Include stabilizing agents (glycerol, specific lipids) in buffers

  • Minimize time between purification steps

• Reconstitution refinement:

  • Total solubilization of phospholipids and proteins in a Triton X-100/octyl glucoside mixture (containing 20 mM octyl glucoside) can lead to threefold stimulation of ATP synthase activity through enhanced activation

  • Optimize lipid composition - negatively charged phospholipids are essential for function

  • Consider adding cholesterol, which can induce a fourfold increase in activity

  • Ensure proper protein orientation during reconstitution (methods achieving 70% correct orientation have been reported)

• Activity enhancement:

  • Optimize buffer conditions (pH, salt concentration)

  • Test different ATP/ADP ratios and Mg2+ concentrations

  • Ensure complete removal of detergents after reconstitution

By systematically addressing these factors, researchers can significantly improve the functional yield of recombinant Bacillus PS3 ATP synthase.

How can researchers distinguish between different conformational states of the ε subunit in experimental settings?

Distinguishing between the inhibitory "up" and permissive "down" conformations of the ε subunit requires specialized experimental approaches:

• Biochemical methods:

  • ATP hydrolysis assays at varying ATP concentrations - Bacillus PS3 shows ATP-dependent inhibition relief at high ATP concentrations (>1 mM)

  • Crosslinking experiments to trap specific conformational states

  • Limited proteolysis - different conformations show distinct digestion patterns

• Structural approaches:

  • Cryo-EM classification to separate particles in different conformational states

  • FRET (Förster Resonance Energy Transfer) using labeled residues to monitor distances between domains

  • EPR (Electron Paramagnetic Resonance) spectroscopy with spin-labeled residues

• Engineered constructs:

  • Introduction of disulfide bonds to lock the ε subunit in specific conformations

  • Truncation constructs lacking portions of the C-terminal domain

  • Point mutations that stabilize either the "up" or "down" conformation

• Data interpretation considerations:

  • ATP concentration dependence is a key indicator - low activity at low ATP (<0.7 mM) and higher activity at high ATP (>1 mM) suggests intact ε regulation

  • Temperature effects - ensure measurements are performed at physiologically relevant temperatures (40°C for Bacillus PS3)

  • Time-dependent measurements to capture dynamic switching between states

These approaches provide complementary information about the regulatory mechanism and can help resolve ambiguities in experimental data.

How do ATP binding properties differ between Bacillus PS3 ATP synthase and other bacterial homologs?

ATP binding properties vary significantly across bacterial ATP synthases despite high sequence similarity:

These differences in ATP binding reflect evolutionary adaptations to different environmental niches and energy requirements . The correlation between sequence variations and binding properties provides valuable insights for protein engineering approaches.

The seemingly minor sequence differences between Bacillus PS3 and Bacillus subtilis that result in dramatically different ATP binding affinities (4 mM vs. 2 mM) highlight the complexity of structure-function relationships and suggest the presence of additional factors beyond the primary ATP binding motif .

What experimental designs can best evaluate the efficiency of Bacillus PS3 ATP synthase in artificial systems?

Evaluation of Bacillus PS3 ATP synthase efficiency in artificial systems requires comprehensive experimental designs:

• Light-driven ATP synthesis measurement:

  • Quantification of ATP production rates under defined illumination conditions

  • Determination of quantum efficiency (ATP molecules produced per photon)

  • Comparison of different reconstitution methods using standardized activity assays (500-800 nmol ATP × min⁻¹ × mg TF0F1⁻¹ represents optimized systems)

• Proton motive force analysis:

  • Measurement of pH gradients using pH-sensitive fluorescent dyes

  • Determination of membrane potential using voltage-sensitive probes

  • Correlation between proton gradient formation and ATP synthesis rates

• System integration assessment:

  • Protein synthesis rates in artificial photosynthetic cells

  • Recycling efficiency - how effectively synthesized components incorporate into the system

  • Long-term stability and self-maintenance capacity

• Comparative performance metrics:

  • Side-by-side comparison with other ATP synthases (mitochondrial, chloroplast)

  • Efficiency under varying conditions (temperature, pH, illumination intensity)

  • Response to inhibitors and regulatory factors

• Energy conversion efficiency calculation:

  • Input energy (light) versus output energy (ATP chemical potential)

  • Identification of rate-limiting steps in the energy conversion process

  • Mathematical modeling of system performance under different conditions

These multifaceted approaches provide a comprehensive understanding of system performance and identify optimization opportunities.

What are promising avenues for engineering Bacillus PS3 ATP synthase for enhanced functionality in synthetic biology applications?

Several promising engineering approaches could enhance Bacillus PS3 ATP synthase functionality:

• Regulatory modifications:

  • Targeted mutations in the ε subunit to alter ATP binding affinity

  • Engineering constructs with modified inhibitory properties

  • Creating variants with altered response to regulatory signals

• Stability enhancements:

  • Further increasing thermostability for industrial applications

  • Improving detergent resistance for easier handling

  • Developing variants with enhanced stability in non-native membrane environments

• Coupling efficiency optimization:

  • Modifications to the a/c subunit interface to enhance proton translocation efficiency

  • Engineering optimized rotor-stator interactions

  • Reducing proton leakage through the F0 portion

• Integration with alternative energy inputs:

  • Coupling ATP synthase to different light-harvesting systems

  • Creating chimeric constructs that respond to alternative energy sources

  • Developing systems that can utilize varied ion gradients (Na+, K+)

• Scalability improvements:

  • Simplifying reconstitution protocols for high-throughput production

  • Developing immobilization strategies for continuous operation

  • Creating self-assembling systems with enhanced yield and reproducibility

These engineering approaches could significantly expand the utility of Bacillus PS3 ATP synthase in synthetic biology applications, artificial photosynthesis, and bioenergy production .

How might advanced structural biology techniques further elucidate the mechanism of Bacillus PS3 ATP synthase?

Emerging structural biology techniques offer exciting opportunities to further understand Bacillus PS3 ATP synthase:

• Time-resolved cryo-EM:

  • Capturing short-lived intermediate states during ATP synthesis/hydrolysis

  • Visualizing conformational changes during rotary catalysis

  • Mapping the complete rotational cycle with millisecond resolution

• Single-molecule techniques:

  • High-speed AFM to directly observe rotational motion

  • Magnetic tweezers to measure torque generation during rotation

  • Single-molecule FRET to track conformational changes in real-time

• Integrative structural approaches:

  • Combining cryo-EM with mass spectrometry to identify post-translational modifications

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions

  • Integrating computational modeling with experimental data for complete mechanistic understanding

• In situ structural studies:

  • Cryo-electron tomography of ATP synthase in native membrane environments

  • Correlative light and electron microscopy to link structure and function

  • Focused ion beam milling combined with cryo-EM for structural studies in cellular context

• Dynamic simulations:

  • Molecular dynamics simulations based on high-resolution structures

  • Quantum mechanical calculations of the proton translocation process

  • Coarse-grained simulations of the complete rotary mechanism