Recombinant Geobacillus stearothermophilus ATP synthase subunit c (atpE)

What is Geobacillus stearothermophilus ATP synthase subunit c (atpE)?

ATP synthase subunit c (atpE) from Geobacillus stearothermophilus is a component of the F₀ sector of F₀F₁-ATP synthase, the enzyme responsible for ATP production. This particular protein is a 72-amino acid polypeptide that functions as part of the membrane-embedded proton channel . The subunit c forms an oligomeric ring structure within the F₀ sector that rotates during ATP synthesis, driven by proton flow through the F₀ complex. In G. stearothermophilus, the protein has been identified with UniProt number P42011 and is sometimes also referred to as ATP synthase F(0) sector subunit c, F-type ATPase subunit c, or lipid-binding protein .

How does G. stearothermophilus ATP synthase differ from other bacterial ATP synthases?

G. stearothermophilus is a thermophilic bacterium with an optimal growth temperature range of 43-76°C . Consequently, its ATP synthase exhibits remarkable thermostability compared to mesophilic counterparts like Escherichia coli. This thermostability makes it particularly valuable for structural and functional studies. While the fundamental mechanism is conserved, G. stearothermophilus ATP synthase has evolved specific adaptations that allow it to maintain structural integrity and function at elevated temperatures. These thermophilic adaptations include more rigid protein conformations, stronger subunit interactions, and specific amino acid compositions that favor stability at high temperatures .

What is the mechanism of ATP synthesis by G. stearothermophilus F₀F₁-ATP synthase?

G. stearothermophilus F₀F₁-ATP synthase functions through a rotational catalytic mechanism. The process begins with proton translocation through the F₀ sector, which drives rotation of the c-subunit oligomeric ring relative to the a-subunit . This rotational force is transferred via the γ subunit to the catalytic F₁ sector, where it induces conformational changes in the αβ pairs, leading to ATP synthesis from ADP and inorganic phosphate (Pi) .

During ATP synthesis, protons move through the a-subunit channel and bind to the glutamic acid residue (E56) of the c-subunits. As the c-ring rotates, the protonated glutamic acid residues move through the hydrophobic membrane environment. When they reach the opposite side of the membrane, protons are released into the cytoplasm, completing the proton translocation process . Importantly, the enzyme also regulates ATP hydrolysis to prevent wasteful ATP consumption, with the ε subunit playing a critical inhibitory role in this regulation .

How do mutations in the c-subunit affect ATP synthase function?

Mutations in the c-subunit can significantly impact ATP synthase function, as demonstrated by studies with the E56D mutation. When glutamic acid at position 56 (E56) was substituted with aspartic acid (D) in one of the c-subunits:

• ATP synthesis activity decreased substantially but was not completely eliminated

• ATP-driven proton pump activity was reduced but partially retained

• The presence of a carboxyl group capable of protonation and deprotonation remained critical for rotation coupled with proton transfer

The effects of mutations become more pronounced with multiple substitutions. When two c-subunits contained the E56D mutation, ATP synthesis activity decreased further. Interestingly, the decrease in activity correlated with the distance between the two mutation sites - as the distance between mutations increased, enzymatic activity declined more significantly .

These findings suggest that subtle structural differences in the proton-binding site, such as the one-methylene-group difference between glutamic acid and aspartic acid side chains, can slow the elementary processes required for driving rotation .

What evidence exists for cooperation among c-subunits in G. stearothermophilus ATP synthase?

Research using genetically fused single-chain c-rings has provided compelling evidence for cooperation among c-subunits in G. stearothermophilus ATP synthase. Key findings include:

• ATP synthesis activity progressively decreased with increasing numbers of E56D mutations

• For double E56D mutations, activity decreased more significantly as the distance between mutation sites increased

• Proton transfer-coupled molecular dynamics simulations reproduced these biochemical findings

Analysis of simulation trajectories revealed that prolonged duration times for proton uptake in two mutated c-subunits can be shared between them. As the distance between mutation sites increases, this time-sharing decreases, explaining the observed reduction in activity .

The table below summarizes the relationship between mutation distance and activity:

These findings demonstrate that functional coupling between neighboring c-subunits is essential for optimal ATP synthase performance .

What expression systems are suitable for producing recombinant G. stearothermophilus ATP synthase subunit c?

For expressing recombinant G. stearothermophilus ATP synthase subunit c, researchers should consider the following expression systems and conditions:

• E. coli Expression System: Due to its versatility and ease of genetic manipulation, E. coli is often the preferred host for expressing recombinant proteins, including thermophilic proteins. For optimal expression of G. stearothermophilus ATP synthase subunit c, researchers should:

  • Use expression vectors with strong, inducible promoters (like T7)

  • Consider codon optimization for E. coli if expression levels are low

  • Express at moderately elevated temperatures (30-37°C) to facilitate proper folding

• Bacillus Expression Systems: Since G. stearothermophilus is a Bacillus-related species, Bacillus subtilis expression systems may provide advantages for proper folding and post-translational modifications.

• Expression Conditions: The expression should be conducted in nutrient-rich broth, supplemented with appropriate antibiotics based on the selection marker. Expression can be induced once the culture reaches mid-log phase .

When working with ATP synthase subunit c, it's crucial to design constructs that maintain the native structural features while adding appropriate tags for purification without disrupting function.

What purification strategies yield the highest purity and activity of G. stearothermophilus ATP synthase subunit c?

To achieve high purity and activity when purifying recombinant G. stearothermophilus ATP synthase subunit c, researchers should implement a multi-step purification strategy:

• Initial Extraction:

  • For membrane proteins like subunit c, use appropriate detergents for solubilization

  • Prepare cell lysates under conditions that preserve protein structure

• Chromatographic Purification:

  • Implement cation-exchange chromatography as a primary purification step

  • Use affinity chromatography if the recombinant protein contains an affinity tag

  • Consider hydrophobic interaction chromatography to exploit the hydrophobic nature of subunit c

• Quality Assessment:

  • Analyze purified fractions using SDS-PAGE to confirm protein purity

  • Confirm identity using mass spectrometry (MS) analysis

  • Verify functionality through biochemical assays relevant to ATP synthase activity

• Storage Conditions:

  • Store in Tris-based buffer with 50% glycerol for optimal stability

  • Store at -20°C or -80°C for extended storage

  • Avoid repeated freeze-thaw cycles

  • For short-term work, maintain working aliquots at 4°C for up to one week

This purification approach has been successfully employed for related proteins from G. stearothermophilus and can be adapted for the ATP synthase subunit c .

How can researchers effectively study c-subunit rotation and proton translocation?

Studying c-subunit rotation and proton translocation in G. stearothermophilus ATP synthase requires specialized techniques:

• Genetic Engineering Approaches:

  • Create genetically fused single-chain c-rings to control the composition of the c-ring

  • Introduce specific mutations (e.g., E56D) in individual c-subunits to study their effects on rotation and proton translocation

  • Develop heteromutated c-rings with various mutation combinations and positions

• Biochemical Activity Assays:

  • Measure ATP synthesis activity to assess the impact of mutations on function

  • Evaluate ATP-driven proton pump activity to study the reverse direction

  • Assess DCCD-sensitive ATP hydrolysis activity (DCCD is a specific inhibitor of the F₀ sector)

• Molecular Dynamics Simulations:

  • Implement proton transfer-coupled molecular dynamics simulations

  • Mimic mutations in silico to reproduce and explain experimental observations

  • Analyze simulation trajectories to understand proton uptake dynamics and cooperation between c-subunits

• Biophysical Techniques:

  • Use cryogenic electron microscopy (cryo-EM) to determine structural conformations

  • Apply single-molecule techniques to directly observe rotation

  • Implement fluorescence-based methods to track conformational changes during activity

These methodologies provide complementary insights into the complex mechanisms of c-subunit rotation and proton translocation in ATP synthase .

How do thermophilic adaptations in G. stearothermophilus ATP synthase contribute to its functional stability?

G. stearothermophilus ATP synthase operates optimally within the growth temperature range of its host organism (43-76°C) , which necessitates specific adaptations for thermostability while maintaining functional flexibility. Several key thermophilic adaptations contribute to this stability:

• Amino Acid Composition:

  • The c-subunit sequence contains a higher proportion of hydrophobic residues that enhance protein-lipid interactions in the membrane at elevated temperatures

  • The presence of alanine-rich regions (MSLGVLAAAIAVGLGALGAG) contributes to helix stability at high temperatures

• Structural Features:

  • More rigid protein conformations that resist thermal denaturation

  • Enhanced subunit-subunit interactions within the c-ring that maintain structural integrity

  • Optimized electrostatic interactions that become stronger at elevated temperatures

• Functional Implications:

  • The thermostable nature of G. stearothermophilus ATP synthase allows it to maintain ATP synthesis capacity at temperatures that would denature mesophilic enzymes

  • Conformational flexibility is preserved despite the increased rigidity, enabling the critical rotational mechanism

  • Proton binding and release kinetics are adapted to operate efficiently at higher temperatures

Understanding these thermophilic adaptations provides valuable insights into protein engineering strategies for enhancing thermostability in biotechnological applications while maintaining functional properties.

What are the key differences in regulatory mechanisms between G. stearothermophilus and other bacterial ATP synthases?

G. stearothermophilus ATP synthase exhibits distinct regulatory mechanisms compared to other bacterial ATP synthases, particularly in how it balances ATP synthesis and hydrolysis activities:

• ε Subunit Regulation:

  • The inhibitory ε subunit in G. stearothermophilus undergoes structural transformations in its C-terminal domain

  • These alterations create a switch between ATP hydrolysis "off" and ATP synthesis "on" states

  • This mechanism helps prevent wasteful ATP consumption while favoring synthesis under appropriate conditions

• Comparative Regulatory Features:

  • In contrast to E. coli, G. stearothermophilus ATP synthase exhibits regulatory adaptations that function optimally at elevated temperatures

  • The thermophilic nature necessitates tighter regulation to prevent futile ATP hydrolysis at high temperatures where chemical reaction rates would naturally increase

  • Structural differences in the catalytic and rotary subunits contribute to altered interactions that influence regulatory outcomes

• Proton Motive Force Sensitivity:

  • G. stearothermophilus ATP synthase appears to have adapted its sensitivity to proton motive force (pmf) to operate efficiently at high temperatures

  • The interaction between pmf and enzyme activity likely differs from that seen in mesophilic bacteria like E. coli

These regulatory differences represent evolutionary adaptations to the thermophilic lifestyle of G. stearothermophilus and offer insights into the diversity of ATP synthase regulation across bacterial species.

How can genetically engineered c-rings inform our understanding of rotational catalysis?

Genetically engineered c-rings, particularly the development of fused single-chain c-rings, have revolutionized our understanding of rotational catalysis in ATP synthases:

• Controlled Compositional Studies:

  • Fused single-chain c-rings allow researchers to precisely control the composition of each c-subunit

  • By introducing specific mutations at defined positions, researchers can study the spatial relationships and cooperative interactions between c-subunits

  • This approach has revealed that the distance between mutation sites significantly impacts functional outcomes

• Mechanistic Insights from Hetero-Mutated c-Rings:

  • Studies with hetero-mutated c-rings have demonstrated that:

    • A single E56D mutation reduces but does not eliminate activity

    • Double E56D mutations further reduce activity, with greater reduction as the distance between mutations increases

    • The presence of a carboxyl group capable of protonation/deprotonation is critical for rotation

• Verification through Complementary Approaches:

  • Biochemical findings from genetically engineered c-rings have been reinforced by molecular dynamics simulations

  • These simulations reveal that prolonged proton uptake times in mutated c-subunits can be shared between subunits

  • The degree of time-sharing decreases as the distance between mutation sites increases, explaining the observed activity patterns

This integrated approach combining genetic engineering with biochemical characterization and computational modeling provides a powerful framework for understanding the complex dynamics of rotational catalysis in ATP synthases.

What parameters should researchers monitor when analyzing G. stearothermophilus ATP synthase activity?

When analyzing G. stearothermophilus ATP synthase activity, researchers should monitor multiple parameters to gain comprehensive insights:

• ATP Synthesis/Hydrolysis Rates:

  • Measure ATP synthesis rates under varying conditions (temperature, pH, substrate concentrations)

  • Quantify ATP hydrolysis rates with and without inhibitors

  • Determine the ratio between synthesis and hydrolysis activities as an indicator of regulatory efficiency

• Proton Translocation Parameters:

  • Monitor proton uptake and release kinetics

  • Measure proton pump activity driven by ATP hydrolysis

  • Calculate the H⁺/ATP ratio under different conditions

• Temperature-Dependent Behavior:

  • Assess activity across the temperature range of 43-76°C

  • Determine temperature optima for different activities

  • Analyze thermostability and inactivation kinetics

• Ion Dependencies:

  • Evaluate the strong dependence on Ca²⁺ ions, which can cause a 10,000-fold decrease in yield when omitted

  • Assess the impact of other ions on activity and stability

• Rotational Dynamics:

  • Measure rotational rates under various conditions

  • Determine the effects of mutations on rotational efficiency

  • Analyze pausing or irregular rotation patterns as indicators of functional impairment

These parameters provide a multi-dimensional profile of ATP synthase function that is essential for understanding both wild-type enzyme behavior and the effects of experimental manipulations.

How should researchers interpret cooperativity data from mutational studies of the c-ring?

Interpreting cooperativity data from c-ring mutational studies requires careful consideration of several factors:

• Positional Effects vs. Cumulative Effects:

  • Distinguish between effects caused by the number of mutations and those caused by the spatial distribution of mutations

  • When activity decreases more as the distance between two mutations increases, this suggests functional coupling between c-subunits rather than simple additive effects

• Correlation with Molecular Simulations:

  • Compare experimental data with results from proton transfer-coupled molecular dynamics simulations

  • When simulations reproduce the experimental trends, this strengthens the interpretation of cooperative mechanisms

  • Analysis of simulation trajectories can reveal molecular details, such as shared prolonged duration times for proton uptake

• Interpretation Framework:

  • Decreased activity with increased distance between mutations suggests that neighboring c-subunits functionally cooperate during rotation

  • The transmission of conformational changes between adjacent c-subunits appears to be an essential component of efficient rotation

  • Time-sharing of proton uptake processes between mutated c-subunits provides a molecular explanation for the observed cooperativity

What computational approaches are most effective for studying c-subunit structure-function relationships?

Several computational approaches have proven valuable for studying c-subunit structure-function relationships in ATP synthase:

• Proton Transfer-Coupled Molecular Dynamics Simulations:

  • These simulations can reproduce key characteristics observed in biochemical experiments

  • They allow analysis of proton uptake duration times and sharing between mutated c-subunits

  • By mimicking mutations in silico, researchers can predict functional outcomes before experimental validation

• Structural Bioinformatics:

  • Sequence analysis and alignment of c-subunits across species reveals conserved features critical for function

  • Comparative modeling of thermophilic vs. mesophilic c-subunits highlights adaptations for thermostability

  • Prediction of protein-protein and protein-lipid interactions informs understanding of c-ring assembly and membrane integration

• Quantum Mechanical/Molecular Mechanical (QM/MM) Approaches:

  • More accurate modeling of proton transfer events requires quantum mechanical calculations

  • QM/MM approaches combine quantum mechanics for the proton transfer site with molecular mechanics for the surrounding protein environment

  • These methods provide insights into the energetics and transition states of proton binding and release events

• Integration with Experimental Data:

  • Computational models should be validated and refined using experimental data

  • Cryo-EM structures of transition states can inform computational models

  • Iterative refinement between computational predictions and experimental validation strengthens both approaches

By employing these computational strategies, researchers can gain deeper insights into the molecular mechanisms underlying c-subunit function in G. stearothermophilus ATP synthase, facilitating both fundamental understanding and potential applications in biotechnology.