Recombinant Nostoc sp. ATP synthase subunit c (atpE)

What is the basic structure of Nostoc sp. ATP synthase subunit c (atpE)?

Nostoc sp. ATP synthase subunit c (atpE) is a small, hydrophobic membrane protein consisting of 81 amino acids. The full amino acid sequence is: MDPLVSAASVLAAALAVGLAAIGPGIGQGNAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALVLLFANPFA . The protein contains primarily hydrophobic regions, consistent with its role as a membrane-embedded component of the F0 sector of ATP synthase. It functions as part of the c-ring structure within the F0 domain, which forms the proton channel across the membrane. The hydrophobic nature of this protein facilitates its integration into the lipid bilayer, where it participates in the rotational mechanism that couples proton translocation to ATP synthesis.

How does atpE contribute to ATP synthase function in cyanobacteria?

The atpE protein forms part of the c-ring structure in the F0 sector of ATP synthase in cyanobacteria like Nostoc sp. This ring plays a crucial role in the rotary mechanism of ATP synthesis by facilitating proton translocation across the thylakoid membrane. Each c-subunit contributes to proton binding sites that, when filled sequentially, cause rotation of the c-ring. This mechanical rotation is coupled to conformational changes in the F1 sector, driving ATP synthesis through oxidative phosphorylation .

In cyanobacteria, the ATP synthase complex operates within the thylakoid membrane system and works in conjunction with photosynthetic electron transport to generate ATP. The complex must also be regulated to prevent wasteful ATP hydrolysis under unfavorable conditions, such as darkness, where a regulatory protein called AtpΘ (previously Norf1) acts as an inhibitor to arrest ATP hydrolysis .

What are the optimal storage and handling conditions for recombinant atpE protein?

Based on manufacturer protocols, recombinant Nostoc sp. ATP synthase subunit c (atpE) protein requires specific handling to maintain stability and function:

Storage conditions:

• Long-term storage: -20°C to -80°C

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

• Avoid repeated freeze-thaw cycles

Reconstitution protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (typically 50%) for long-term storage

• Aliquot to minimize freeze-thaw cycles

Buffer composition:

• Typically supplied in Tris/PBS-based buffer with 6% Trehalose, pH 8.0

• Supplied as lyophilized powder with >90% purity as determined by SDS-PAGE

These handling procedures are critical for maintaining protein integrity, particularly for membrane proteins like atpE that tend to aggregate when improperly handled.

What methods are most effective for analyzing atpE interactions with other ATP synthase components?

Several complementary approaches have proven effective for studying atpE interactions:

Membrane fractionation studies:

• Differential centrifugation to isolate membrane fractions

• Sucrose gradient ultracentrifugation for purification of membrane complexes

• Western blotting with anti-atpE antibodies to confirm protein localization

Protein-protein interaction analyses:

• Immunoprecipitation coupled with mass spectrometry to identify interaction partners

• Far Western blotting to detect direct protein-protein interactions

• Blue native PAGE to preserve native protein complexes

• Fluorescence resonance energy transfer (FRET) with tagged proteins to examine proximity in situ

Functional assays:

• ATP hydrolysis measurements in isolated membrane fractions

• Proton translocation assays using pH-sensitive fluorescent dyes

• Site-directed mutagenesis to identify critical residues for interaction

Studies with cyanobacterial ATP synthase components have successfully used GFP fusion proteins to track membrane targeting, combined with immunoprecipitation and mass spectrometry to identify interacting proteins . These approaches revealed that regulatory proteins like AtpΘ interact with ATP synthase complexes under specific conditions to modulate function.

How does the expression of atpE vary under different environmental conditions in Nostoc species?

The expression of ATP synthase components, including atpE, in Nostoc and related cyanobacteria shows significant environmental regulation:

Light/dark transitions:
Northern hybridization analyses have demonstrated that expression of ATP synthase-related genes is differentially regulated between light and dark conditions. For example, the atpT gene (encoding the ATP synthase inhibitory factor AtpΘ) shows significantly increased expression after 6 hours in darkness compared to light conditions in several cyanobacteria, including Nostoc 7120 . This suggests a coordinated regulation mechanism to prevent wasteful ATP hydrolysis during periods of limited energy availability.

Time course of expression:
In Nostoc 7120, detailed time course studies show that expression of ATP synthase regulatory components can change rapidly:

• Initial induction within 15 minutes of darkness

• Maximum expression reached at 45 minutes after transfer to darkness

Stress responses:
Under salt stress conditions, ATP synthase activity may be affected by cellular adaptations. Studies with the related cyanobacterium Anabaena PCC 7120 show that osmolytes like glycinebetaine (GB) can interact with membrane protein complexes to maintain and even stimulate their activity under stress conditions . While this has been specifically demonstrated for nitrate transporters, similar interactions could potentially affect ATP synthase function and regulation.

These regulatory patterns indicate sophisticated control mechanisms that synchronize ATP synthase activity with environmental conditions and cellular energy requirements.

What is the evolutionary conservation of atpE across different cyanobacterial species?

Phylogenetic analyses of ATP synthase components reveal significant evolutionary patterns:

Sequence conservation:
The atpE subunit is highly conserved among cyanobacteria, reflecting its critical role in the fundamental process of ATP synthesis. Comparison of amino acid sequences shows high conservation, particularly in the transmembrane regions and proton-binding sites.

Phylogenetic relationships:
Analysis of related ATP-synthase components has shown that:

• Nostoc PCC 7120 is very closely related to Anabaena variabilis ATCC 29413, Anabaena sp. 4-3, and Anabaena sp. CA = ATCC 33047

• Nostoc spp. NIES-3756 and PCC 7524 often appear in the same subclade, suggesting a close evolutionary relationship

The high degree of conservation in ATP synthase components reflects the essential nature of this enzyme complex across cyanobacterial lineages, while variations in specific residues may relate to adaptations to different ecological niches.

How do the structural features of atpE relate to its role in proton translocation?

The structural features of atpE are intimately connected to its function in proton translocation:

Hydrophobic domains:
The protein sequence (MDPLVSAASVLAAALAVGLAAIGPGIGQGNAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALVLLFANPFA) reveals distinct hydrophobic regions that facilitate its embedding in the lipid bilayer . These hydrophobic segments form transmembrane α-helices that span the membrane.

Functional motifs:
Each c-subunit contains a conserved acidic residue (typically aspartate or glutamate) that is essential for proton binding and translocation. This residue undergoes protonation/deprotonation cycles as part of the proton translocation mechanism.

Oligomeric arrangement:
Multiple c-subunits assemble into a ring structure (typically 8-15 subunits depending on the species), creating a central pore. The precise stoichiometry of the c-ring affects the bioenergetic efficiency of ATP synthesis, as it determines the H⁺/ATP ratio.

Interaction interfaces:
Specific regions of the c-subunit interact with other components of the ATP synthase complex:

• The polar loop regions interact with the a-subunit to form the proton translocation pathway

• The c-ring interacts with the γ and ε subunits of the central stalk, coupling proton flow to rotation

These structural features enable the c-ring to function as a rotary motor driven by the proton motive force, converting electrochemical energy into mechanical rotation that drives ATP synthesis.

What are the optimal conditions for expressing recombinant Nostoc sp. atpE in E. coli systems?

Expressing membrane proteins like atpE presents specific challenges that can be addressed through optimized protocols:

Expression system selection:

• E. coli BL21(DE3) or C41(DE3)/C43(DE3) strains are preferred for membrane protein expression

• C41/C43 strains, derived from BL21, are specifically engineered to handle toxic membrane proteins

Vector considerations:

• pET vectors with T7 promoter systems offer controllable expression

• Addition of N-terminal His-tag facilitates purification while minimizing interference with membrane insertion

• Fusion partners like MBP or SUMO can improve solubility

Culture conditions:

• Lower temperatures (16-25°C) after induction to slow expression and improve folding

• Lower IPTG concentrations (0.1-0.5 mM) for gradual induction

• Rich media (TB or 2YT) to support cell growth with membrane protein burden

• Supplementation with additional phospholipids can improve yield

Induction protocol:

• Grow cultures to mid-log phase (OD₆₀₀ = 0.6-0.8)

• Reduce temperature to 18°C

• Induce with 0.2-0.5 mM IPTG

• Express for 16-20 hours

Extraction and purification:

• Cell disruption by sonication or high-pressure homogenization

• Membrane fraction isolation by ultracentrifugation

• Solubilization with mild detergents (DDM, LDAO, or C₁₂E₈)

• IMAC purification using Ni-NTA resin

• Further purification by size exclusion chromatography

The commercially available recombinant Nostoc sp. ATP synthase subunit c (atpE) is produced using E. coli expression systems with N-terminal His-tags, resulting in >90% purity as determined by SDS-PAGE .

How can researchers effectively reconstitute atpE into proteoliposomes for functional studies?

Reconstitution of atpE into proteoliposomes is essential for functional studies of proton translocation and ATP synthesis:

Lipid composition optimization:

• E. coli polar lipid extract (70%) with DOPC (30%) mimics bacterial membranes

• Alternative: DOPC/DOPE/DOPG (7:2:1) mixture

• Cholesterol (0-20%) can be added to modify membrane properties

Reconstitution protocol:

• Preparation of lipid vesicles:

  • Dissolve lipids in chloroform

  • Evaporate solvent under nitrogen stream

  • Rehydrate lipid film in reconstitution buffer (typically 10 mM HEPES, 100 mM KCl, pH 7.4)

  • Sonicate or extrude to form unilamellar vesicles

• Protein incorporation:

  • Mix purified atpE protein (in detergent) with preformed liposomes

  • Detergent removal methods:

    • Bio-Beads SM-2 adsorption (gentle, gradual removal)

    • Dialysis (slower, gentler process)

    • Gel filtration (rapid removal)

• Functional validation:

  • Freeze-fracture electron microscopy to confirm protein incorporation

  • Dynamic light scattering to assess vesicle size distribution

  • Fluorescence-based assays with pH-sensitive dyes (ACMA or pyranine) to measure proton translocation

Critical parameters for successful reconstitution:

• Protein-to-lipid ratio (typically 1:50 to 1:200 w/w)

• Detergent concentration during mixing (above CMC but below solubilizing concentration for liposomes)

• Rate of detergent removal (gradual removal preserves vesicle integrity)

• Buffer ionic strength and pH

For complete ATP synthase functional studies, co-reconstitution of atpE with other subunits of the F₀ and F₁ sectors is necessary to reconstitute full ATP synthesis activity.

How does atpE interact with regulatory proteins like AtpΘ in cyanobacterial ATP synthase?

Recent research has uncovered sophisticated regulatory mechanisms for ATP synthase in cyanobacteria:

Discovery of AtpΘ as a regulatory factor:
A protein previously called Norf1 (novel ORF1) has been identified as an inhibitory factor for ATP synthase in cyanobacteria, now renamed AtpΘ (ATP synthase inhibiTory factor, gene atpT). This protein is recruited under unfavorable conditions to prevent wasteful ATP hydrolysis .

Interaction mechanism:

• AtpΘ is a soluble protein that targets to the thylakoid membrane

• Membrane fractionation experiments and GFP fusion studies confirmed its membrane localization

• Immunoprecipitation followed by mass spectrometry revealed specific interactions with ATP synthase subunits

• Far Western blot analysis provided additional evidence for these interactions

Functional impact:
Measurements of ATP hydrolysis in isolated membrane fractions and purified ATP synthase complexes demonstrated that AtpΘ inhibits ATP hydrolysis activity. This inhibition is particularly important during energy-limited conditions like darkness, preventing the reversal of ATP synthase that would wastefully consume ATP .

Expression patterns:
Northern hybridization analyses showed that atpT gene expression is significantly increased after 6 hours in darkness compared to light conditions in multiple cyanobacterial species, including Nostoc 7120. Time course studies revealed rapid induction within 15 minutes of darkness, reaching maximum expression after 45 minutes .

These findings highlight the sophisticated regulatory mechanisms that have evolved to control ATP synthase activity in response to changing environmental conditions in cyanobacteria.

What role might atpE play in adaptation to environmental stresses in Nostoc species?

The ATP synthase complex, including the atpE subunit, appears to be involved in stress adaptation mechanisms in cyanobacteria:

Salt stress responses:
Studies with the related cyanobacterium Anabaena PCC 7120 have shown that membrane protein complexes can be protected and even stimulated under salt stress by compatible solutes like glycinebetaine (GB). While this has been directly demonstrated for nitrate transporters, it suggests a broader mechanism that might also apply to ATP synthase .

Interaction with protective molecules:
Molecular interaction studies have revealed that:

• Glycinebetaine interacts hydrophobically with membrane proteins

• These interactions can show higher affinity than the natural substrates

• Such interactions maintain or enhance protein function under stress conditions

Potential adaptation mechanisms:

Research implications:
These findings suggest that engineering approaches targeting the interaction between ATP synthase components and protective molecules could potentially enhance stress tolerance in biotechnologically important cyanobacteria.

Further research is needed to directly characterize the interactions between atpE and stress protectants like glycinebetaine, and to determine how these interactions affect ATP synthase function under various stress conditions.

What are common challenges in working with recombinant atpE and how can they be addressed?

Membrane proteins like atpE present several technical challenges that researchers should anticipate:

Solubility and aggregation issues:

• Problem: Hydrophobic membrane proteins tend to aggregate during expression and purification

• Solution: Use mild detergents like DDM, LDAO, or digitonin; consider adding lipids during purification; work at lower temperatures (4°C); avoid concentrating above 5 mg/mL

Purification difficulties:

• Problem: Low yield and purity from standard protocols

• Solution: Optimize detergent type and concentration; use two-step purification (IMAC followed by size exclusion); consider affinity tags positioned to not interfere with membrane domains

Protein stability:

• Problem: Rapid degradation during storage and handling

• Solution: Add glycerol (50%) for storage; store in aliquots at -80°C; avoid repeated freeze-thaw cycles; store working aliquots at 4°C for no more than one week

Functional reconstitution challenges:

• Problem: Loss of activity during reconstitution into liposomes

• Solution: Optimize lipid composition; use gentle detergent removal methods; validate incorporation using multiple methods

Expression toxicity:

• Problem: Toxic effects on host cells during overexpression

• Solution: Use specialized strains (C41/C43); reduce induction temperature and IPTG concentration; consider tightly regulated expression systems

Protein-specific considerations for atpE:

• The small size (81 amino acids) and highly hydrophobic nature of atpE make it particularly challenging

• Multiple transmembrane domains require careful consideration in detergent selection

• The native oligomeric state (c-ring) should be considered when evaluating purification results

How can researchers validate the structural integrity and functionality of purified recombinant atpE?

Multiple complementary approaches should be used to validate recombinant atpE quality:

Structural integrity assessment:

• SDS-PAGE analysis: Confirms correct molecular weight and >90% purity

• Circular dichroism (CD) spectroscopy: Verifies secondary structure content (primarily α-helical for atpE)

• Thermal stability assays: Monitors protein unfolding using dyes like SYPRO Orange in differential scanning fluorimetry

Functional validation:

• Proton translocation assays: Using pH-sensitive dyes in reconstituted proteoliposomes

• ATP synthesis/hydrolysis measurements: When co-reconstituted with other ATP synthase components

• Oligomerization analysis: Size exclusion chromatography or native PAGE to assess c-ring formation

Binding studies:

• Interaction with known binding partners: Surface plasmon resonance (SPR) or microscale thermophoresis (MST) with other ATP synthase subunits

• Lipid binding assays: Using labeled lipids or membrane mimetics

Quality control parameters and benchmarks:

• Expected purity: >90% by SDS-PAGE

• Secondary structure: High α-helical content by CD spectroscopy

• Thermostability: Tm should be consistent batch-to-batch

• Functional activity: Reproducible proton translocation rates when properly reconstituted

By applying these multiple validation approaches, researchers can ensure that their recombinant atpE protein maintains native-like properties suitable for downstream structural and functional studies.

What emerging techniques might advance our understanding of atpE structure and function?

Several cutting-edge methodologies hold promise for deeper insights into atpE biology:

Cryo-electron microscopy advancements:

• Recent technical improvements in single-particle cryo-EM now allow near-atomic resolution of membrane protein complexes

• Application to ATP synthase c-rings could reveal precise structural arrangements and conformational changes during proton translocation

• Time-resolved cryo-EM might capture intermediate states during the rotational cycle

Integrative structural biology approaches:

• Combining X-ray crystallography, NMR, and computational modeling to generate comprehensive structural models

• Cross-linking mass spectrometry to map interaction interfaces between atpE and other ATP synthase components

• Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions during function

Advanced microscopy techniques:

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

• High-speed atomic force microscopy to visualize ATP synthase rotation

• Super-resolution microscopy to map ATP synthase distribution and dynamics in native membranes

Computational methods:

• Molecular dynamics simulations of c-ring rotation and proton translocation

• Machine learning approaches to predict functional impacts of sequence variations

• Systems biology modeling to understand ATP synthase regulation in the context of cellular metabolism

These emerging approaches, particularly when used in combination, promise to reveal new aspects of atpE function that have remained elusive with traditional techniques.

How might understanding atpE function contribute to biotechnological applications in cyanobacteria?

Knowledge of ATP synthase components like atpE could enable several biotechnological advances:

Bioenergetic optimization for bioproduction:

• Engineering ATP synthase components to optimize the H⁺/ATP ratio could increase energy efficiency in biotechnologically relevant cyanobacteria

• Modulating ATP synthase regulation through modification of inhibitory factors like AtpΘ could potentially enhance growth rates under specific cultivation conditions

• Understanding how environmental stresses affect ATP synthase function could lead to strains with improved stress tolerance

Synthetic biology applications:

• Engineered c-rings with modified stoichiometry could create strains with altered bioenergetic properties

• ATP synthase components could be repurposed as scaffolds for artificial bionanomachines

• Understanding the molecular interactions between protective molecules like glycinebetaine and membrane proteins could inform strategies for engineering enhanced stress tolerance

Biomimetic energy conversion:

• Insights from cyanobacterial ATP synthase could inspire artificial nanomotors or energy conversion devices

• The principles of proton-driven rotation could inform development of synthetic molecular motors

Environmental biotechnology:

• Optimized ATP synthase function could enhance cyanobacterial growth for bioremediation applications

• Improved bioenergetics could boost production of value-added compounds from waste streams

These applications highlight the potential translational value of fundamental research on atpE and other ATP synthase components in cyanobacteria.