Recombinant Methanosarcina mazei V-type ATP synthase subunit E (atpE)

What is the biological role of subunit E in archaeal ATP synthases?

Subunit E of the Methanosarcina mazei V-type ATP synthase functions as a critical component of the peripheral stalk that connects the A₁ and A₀ sections of the ATP synthase complex. This peripheral stalk is essential for the enzyme's rotary mechanism, as it prevents the catalytic A₁ sector from rotating with the central rotor during ATP synthesis or hydrolysis. Subunit E specifically interacts with other peripheral stalk components, particularly subunit H, to maintain the structural integrity of the enzyme complex . Unlike the bacterial F-type ATP synthases, the archaeal A-type ATP synthases have unique structural and functional properties that allow them to operate efficiently in extreme environments where many archaea thrive.

What are the recommended storage conditions for recombinant atpE?

For optimal stability and activity, recombinant M. mazei atpE should be stored according to these guidelines:

• Liquid form: Store at -20°C/-80°C with a typical shelf life of 6 months

• Lyophilized form: Store at -20°C/-80°C with an extended shelf life of 12 months

• Working aliquots: Store at 4°C for up to one week

• For long-term storage: Reconstitute to 0.1-1.0 mg/mL and add glycerol to a final concentration of 5-50% (default recommendation is 50%)

Repeated freeze-thaw cycles should be avoided as they can compromise protein integrity and activity. Brief centrifugation prior to opening vials is recommended to bring contents to the bottom .

How does the interaction between subunit E and other ATP synthase components contribute to enzyme function?

Structural and functional studies reveal that subunit E forms essential interactions with other components of the archaeal ATP synthase, particularly with subunit H. NMR titration experiments with related archaeal ATP synthases have shown that the N-terminal domain of subunit E (residues E1-100) interacts specifically with the N-terminal region of subunit H, with particular involvement of residues E41-60 . This interaction has been confirmed by fluorescence correlation spectroscopy.

The protein-protein interaction network within the ATP synthase complex is critical for:

• Maintaining the structural integrity of the peripheral stalk

• Preventing co-rotation of the A₁ catalytic sector with the central rotor during ATP synthesis

• Ensuring efficient energy coupling between ion translocation and ATP synthesis/hydrolysis

What methodological approaches are most effective for studying atpE interactions?

Based on successful research with archaeal ATP synthase components, the following methodological approaches are recommended for studying atpE interactions:

NMR Spectroscopy

NMR titration experiments using ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectroscopy have proven effective for mapping protein-protein interactions between ATP synthase subunits. This approach allows identification of specific residues involved in subunit binding . For atpE studies, this would involve:

• Expression and purification of ¹⁵N-labeled recombinant atpE

• Preparation of potential binding partners (other ATP synthase subunits)

• Titration experiments monitoring chemical shift perturbations in HSQC spectra

Fluorescence Correlation Spectroscopy (FCS)

FCS provides complementary data to validate interactions identified by NMR. This technique has successfully confirmed the interaction between N-terminal domains of subunits E and H in archaeal ATP synthases .

Enzymatic Activity Assays

Substrate-dependent ATP hydrolysis experiments can measure the impact of subunit interactions on enzymatic activity. For example, studies with M. mazei subunit F demonstrated significant increases in ATP hydrolysis rates when added to the A₃B₃D complex . Similar approaches could elucidate atpE's functional contributions.

How do the structural features of atpE affect its function in the ATP synthase complex?

The structural organization of atpE contains critical features that determine its function:

• N-terminal domain (approximately residues 1-100): Involved in protein-protein interactions, particularly with subunit H

• C-terminal domain (approximately residues 101-183): May contribute to stability and positioning within the ATP synthase complex

The functional significance of these domains is evidenced by studies of related subunits in archaeal ATP synthases, where deletions of terminal regions significantly impact activity. For example, in subunit F of M. mazei, deletions of either N- or C-termini abolished the ATP hydrolysis activation effect . By analogy, specific regions of atpE likely play crucial roles in maintaining proper assembly and function of the ATP synthase complex.

What expression systems are optimal for producing functional recombinant atpE?

Based on successful recombinant protein production strategies for archaeal proteins:

For optimal expression in E. coli systems:

• Use a vector with an inducible promoter (T7 or similar)

• Transform into a host strain designed for protein expression (BL21(DE3) or derivatives)

• Consider expressing with a fusion tag (His-tag is commonly used)

• Optimize induction conditions (temperature, inducer concentration, duration)

What purification strategies yield the highest purity and activity of recombinant atpE?

A multi-step purification strategy is recommended:

• Initial capture: Affinity chromatography using Ni-NTA resin for His-tagged atpE

• Intermediate purification: Ion exchange chromatography to separate based on charge properties

• Polishing: Size exclusion chromatography to achieve high purity (>85% as measured by SDS-PAGE)

Critical purification parameters:

• Buffer composition: Typically 20-50 mM phosphate or Tris buffer, pH 7.5-8.0, with 100-300 mM NaCl

• Use of reducing agents: Include 1-5 mM DTT or β-mercaptoethanol to prevent oxidation

• Protease inhibitors: Add during initial lysis to prevent degradation

How can researchers effectively study atpE interactions with other ATP synthase components?

A systematic approach to studying atpE interactions should include:

• Domain mapping: Express and purify individual domains of atpE (N-terminal and C-terminal) to identify specific interaction regions

• Co-immunoprecipitation assays: Use antibodies against atpE to pull down interaction partners

• Surface plasmon resonance: Quantify binding affinities between atpE and other subunits

• Reconstitution experiments: Assess the functional impact of atpE on ATP hydrolysis/synthesis using in vitro reconstituted subcomplexes

Previous studies of archaeal ATP synthase subunits have successfully employed NMR titration experiments to map specific residue interactions. For example, NMR studies of subunit H interaction with subunit E identified specific amino acids involved in binding (Met1-6, Lys10, Glu11, Ala15, Val20, and Glu24 of subunit H) . Similar approaches could elucidate the interaction landscape of M. mazei atpE.

How does M. mazei atpE compare to homologous proteins in other archaea and bacteria?

Archaeal ATP synthases bridge the evolutionary gap between bacterial F-type and eukaryotic V-type ATP synthases. Key comparative aspects include:

• Structural comparisons: The peripheral stalk architecture in archaeal A-type ATP synthases (including subunit E) shares more similarities with eukaryotic V-type ATP synthases than with bacterial F-type enzymes

• Functional conservation: Despite structural differences, the fundamental mechanistic principles of rotary catalysis are preserved across all domains of life

• Subunit composition: Archaeal ATP synthases contain subunits not found in bacterial counterparts

Studies with hybrid complexes have demonstrated functional compatibility between some components. For instance, hybrid formation of the A₃B₃D complex with subunit F from eukaryotic V-ATPase (Saccharomyces cerevisiae) or subunit ε from bacterial F-ATP synthase (Mycobacterium tuberculosis) showed that archaeal and eukaryotic subunits share important functions in ATP hydrolysis .

What insights can be gained from studying atpE regarding the evolution of energy conservation mechanisms?

The study of archaeal ATP synthase components like atpE provides valuable insights into the evolution of cellular energy conversion systems:

• Archaeal A-type ATP synthases represent an evolutionary intermediate between bacterial F-type and eukaryotic V-type ATP synthases

• Structural and functional analysis of atpE can help reconstruct the evolutionary path of ATP synthases

• Studies of extremophilic archaea like Methanosarcina mazei reveal adaptations that allow these energy conversion systems to function under harsh conditions

What are the major technical challenges in working with archaeal ATP synthase components?

Researchers face several challenges when working with archaeal ATP synthase components like atpE:

• Protein stability: Maintaining the native structure and function during purification and analysis

• Reconstitution of complex interactions: Accurately recreating the multisubunit ATP synthase complex in vitro

• Species-specific adaptations: Accounting for the unique properties of proteins from extremophilic organisms

• Structural characterization: Obtaining high-resolution structural data for complete ATP synthase complexes

What emerging technologies might enhance our understanding of atpE function?

Several cutting-edge approaches show promise for advancing our understanding of atpE and archaeal ATP synthases:

• Cryo-electron microscopy: For high-resolution structural characterization of the complete ATP synthase complex

• Single-molecule techniques: To observe conformational changes during the catalytic cycle

• Native mass spectrometry: To analyze subunit interactions and complex assembly

• Molecular dynamics simulations: To model the behavior of atpE within the ATP synthase complex

• CRISPR-based genome editing in archaeal systems: For in vivo functional studies