Recombinant Serratia proteamaculans ATP synthase subunit c (atpE)

What is Serratia proteamaculans ATP synthase subunit c (atpE) and what is its functional significance?

ATP synthase subunit c (atpE) is a critical component of the F₀ sector of F₀F₁-ATP synthase in Serratia proteamaculans. This small hydrophobic protein (79 amino acids) forms an oligomeric ring structure in the membrane that functions in proton translocation across the membrane. The amino acid sequence of S. proteamaculans atpE is MENLSMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . The c-subunit contains essential carboxyl groups (typically glutamic acid residues) that undergo protonation and deprotonation cycles during rotational catalysis, driving ATP synthesis. This proton-binding site is critical for the energy transduction mechanism that couples proton translocation to ATP synthesis through rotational motion of the c-ring relative to the a-subunit .

How is recombinant S. proteamaculans atpE typically expressed and purified?

For expression of recombinant S. proteamaculans atpE, the protein is typically produced in E. coli expression systems with an N-terminal His-tag to facilitate purification . The methodological approach involves:

• Cloning the atpE gene (1-79 amino acids) into an appropriate expression vector

• Transformation into E. coli expression host strains

• Induction of protein expression under optimized conditions

• Cell lysis and membrane isolation

• Solubilization using appropriate detergents

• Purification via immobilized metal affinity chromatography (IMAC)

• Further purification steps (size exclusion chromatography if needed)

• Final preparation as a lyophilized powder

The purified protein typically achieves >90% purity as determined by SDS-PAGE analysis . For reconstitution, the lyophilized protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and glycerol (final concentration 5-50%) should be added for long-term storage at -20°C/-80°C .

What structural features characterize ATP synthase subunit c and how do they relate to function?

The ATP synthase subunit c exhibits several key structural features that are essential to its function:

The c-subunit adopts a hairpin-like structure with two transmembrane α-helices connected by a short loop region. The critical glutamic acid residue is positioned such that it can alternatively face either the lipid bilayer (protonated state) or the a-subunit interface (where deprotonation occurs) . This structural arrangement enables the c-ring to function as a proton-driven molecular rotor that drives ATP synthesis through conformational coupling to the F₁ sector.

How do mutations in ATP synthase subunit c affect proton translocation and ATP synthesis?

Mutations in the c-subunit, particularly at the critical glutamic acid residue involved in proton binding, significantly impact ATP synthase function. Studies with Bacillus PS3 ATP synthase demonstrate that replacing the glutamic acid with aspartic acid (E56D) reduces both ATP synthesis and proton pump activities . This reduction occurs because:

• The E56D mutation alters the pKa of the proton-binding site

• The shorter side chain of aspartic acid changes the geometry of the proton binding pocket

• These changes affect the kinetics of proton uptake and release

Interestingly, while the E56D mutation reduces activity, it does not completely eliminate it, indicating that the carboxyl group's protonation/deprotonation capability is essential but that optimal function depends on precise structural positioning . In contrast, the E56Q mutation (replacing glutamic acid with glutamine) completely abolishes ATP synthesis and proton pump activity, confirming that a protonatable carboxyl group is absolutely required for function .

The effects of mutations are quantifiable through activity assays:

The impact of double E56D mutations varies depending on the distance between the mutated c-subunits, revealing cooperation among c-subunits in the rotational mechanism .

What methods are used to investigate cooperation among c-subunits in ATP synthase function?

Investigating cooperation among c-subunits requires sophisticated experimental approaches that allow controlled manipulation of individual subunits within the c-ring. Key methodological approaches include:

• Genetically fused single-chain c-ring construction: This approach involves creating a construct where multiple c-subunits are fused into a single polypeptide chain, allowing site-specific mutations in defined positions. In Bacillus PS3 studies, researchers fused 10 copies of the c-subunit into a single polypeptide (c₁₀) and demonstrated that this construct retained proton-coupled ATP synthesis/hydrolysis activity .

• Site-directed mutagenesis of specific c-subunits: Once the single-chain construct is created, specific mutations (e.g., E56D) can be introduced at defined positions to study positional effects on function. This approach enabled researchers to create six mutant ATP synthases harboring one or two E56D mutations at different positions within the c-ring .

• ATP synthesis and proton pump activity assays: These functional assays quantify the impact of mutations on ATP synthase activity. Studies showed that activity decreased as the distance between two E56D mutations increased, providing evidence for cooperative interactions among c-subunits .

• Proton transfer-coupled molecular dynamics simulations: Computational approaches complement biochemical data by modeling proton transfer events at the atomic level. Simulations revealed that prolonged proton uptake in mutated c-subunits can be shared between subunits, with the degree of time-sharing decreasing as the distance between mutations increases .

These approaches collectively demonstrated that at least three c-subunits at the a/c interface cooperate during c-ring rotation, with optimal activity requiring proper coordination of proton transfer events across multiple subunits .

How can recombinant S. proteamaculans atpE be used for structural studies of ATP synthase?

Recombinant S. proteamaculans atpE provides valuable material for structural investigations using various biophysical techniques:

• X-ray crystallography: Purified c-subunits can be crystallized alone or as part of the c-ring to determine high-resolution structures. This requires:

  • High-purity protein (>95%)

  • Appropriate detergent conditions for maintaining structural integrity

  • Screening of crystallization conditions (pH, temperature, precipitants)

  • Structure determination using molecular replacement or experimental phasing

• Cryo-electron microscopy (cryo-EM): Increasingly used for membrane protein structure determination, requiring:

  • Sample preparation in detergent micelles or nanodiscs

  • Vitrification on EM grids

  • High-resolution image acquisition

  • Computational image processing and 3D reconstruction

• NMR spectroscopy: Useful for studying dynamics and protonation states of c-subunits:

  • Isotopic labeling (¹⁵N, ¹³C, ²H) of recombinant protein

  • Sample preparation in detergent micelles or lipid bilayers

  • Multi-dimensional NMR experiments

  • Structure determination and dynamics analysis

• Cross-linking mass spectrometry: For mapping interactions between c-subunits and with other ATP synthase components:

  • Chemical or photo-crosslinking of purified complexes

  • Proteolytic digestion

  • MS/MS analysis to identify crosslinked peptides

  • Structural modeling based on distance constraints

These approaches provide complementary information about c-subunit structure, assembly, and dynamics that can be integrated into comprehensive structural models of ATP synthase function.

What are optimal conditions for expressing functional recombinant S. proteamaculans atpE?

Optimizing expression conditions is critical for obtaining high yields of properly folded, functional atpE protein:

Additional considerations include:

• Addition of membrane-stabilizing agents (glycerol 5-10%)

• Supplementation with appropriate metal ions if needed for stability

• Monitoring expression using Western blot analysis with anti-His antibodies

• Assessing membrane integration through fractionation experiments

Researchers should optimize these parameters for their specific expression construct and experimental goals .

How can researchers troubleshoot issues with recombinant ATP synthase subunit c expression and purification?

Common challenges in atpE expression and purification include low yields, protein aggregation, and loss of functional properties. Troubleshooting approaches include:

• Low expression levels:

  • Optimize codon usage for expression host

  • Try different promoter systems (T7, tac, ara)

  • Test alternative signal sequences or fusion partners

  • Screen multiple E. coli strains (BL21, C41/C43, Rosetta)

• Protein aggregation/inclusion bodies:

  • Reduce expression temperature (16-20°C)

  • Decrease inducer concentration

  • Co-express with chaperones (GroEL/ES, DnaK/J)

  • Include mild solubilizing agents in lysis buffer

• Poor membrane integration:

  • Optimize membrane targeting sequence

  • Use specialized strains with enhanced membrane capacity

  • Adjust lipid composition of expression host

  • Consider cell-free expression systems with supplied membranes

• Purification challenges:

  • Screen detergents for optimal solubilization (DDM, LDAO, C12E8)

  • Include stabilizing agents (glycerol, specific lipids)

  • Optimize imidazole concentrations to reduce non-specific binding

  • Consider on-column refolding for proteins in inclusion bodies

• Activity loss during storage:

  • Avoid repeated freeze-thaw cycles

  • Store small working aliquots at 4°C for up to one week

  • Use trehalose (6%) in storage buffer for lyophilized samples

  • For long-term storage, include 50% glycerol and store at -20°C/-80°C

Systematic optimization of these parameters can significantly improve the yield and quality of recombinant atpE protein preparations.

What are key considerations when designing site-directed mutagenesis experiments for ATP synthase subunit c?

When designing mutagenesis studies with ATP synthase subunit c, researchers should consider:

These considerations can guide the design of informative mutagenesis experiments that provide mechanistic insights into ATP synthase function, as demonstrated in studies with Bacillus PS3 ATP synthase .