Recombinant Trachelium caeruleum ATP synthase subunit c, chloroplastic (atpH)

What is the basic structure of Trachelium caeruleum ATP synthase subunit c?

Recombinant Full Length Trachelium caeruleum ATP synthase subunit c (atpH) is a 81-amino acid protein with the sequence: MNPLISAASVIAAGLAVGLASIGPGIGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV. It is part of the chloroplastic ATP synthase complex, specifically located in the F0 sector that facilitates proton translocation across membranes. The protein contains a hydrophobic region that integrates into the membrane and is critical for forming the c-ring structure of the ATP synthase complex .

How does the chloroplastic atpH differ from mitochondrial ATP synthase subunit c?

While both proteins serve similar functions in their respective ATP synthase complexes, there are several key differences:

These differences reflect evolutionary adaptations to the distinct environments and energy-coupling mechanisms in chloroplasts versus mitochondria .

What expression systems are optimal for recombinant production of Trachelium caeruleum atpH?

E. coli is the predominant expression system for recombinant T. caeruleum atpH production, as demonstrated in current protocols. The protein is typically expressed with an N-terminal His-tag to facilitate purification. When expressing this highly hydrophobic membrane protein, several considerations must be addressed:

• Expression vector selection: pET-based vectors with strong T7 promoters often yield good results for membrane proteins

• Host strain optimization: C41(DE3) or C43(DE3) strains, derivatives of BL21(DE3), show improved tolerance for membrane protein expression

• Induction conditions: Lower temperatures (16-25°C) and reduced IPTG concentrations (0.1-0.5 mM) tend to improve proper folding

• Membrane extraction: Specialized detergents like n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) are required for efficient solubilization

These approaches help overcome the challenges inherent in expressing membrane proteins while maintaining structural integrity .

What purification strategies yield highest purity and activity for recombinant atpH?

Purification of recombinant His-tagged T. caeruleum atpH typically follows a multi-step process:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co2+-based resins with imidazole gradient elution

• Secondary purification: Size exclusion chromatography to separate monomeric from oligomeric forms

• Buffer optimization: Maintaining appropriate detergent concentrations above critical micelle concentration throughout purification

• Quality assessment: SDS-PAGE analysis confirming >90% purity, with additional verification through Western blotting

• Activity preservation: Addition of phospholipids (often a mixture of POPC/POPE at 3:1 ratio) to stabilize the protein

The final product is often lyophilized with 6% trehalose in Tris/PBS-based buffer at pH 8.0 to maintain stability during storage. Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage at -20°C/-80°C .

How can researchers assess the functional integrity of purified recombinant atpH?

Multiple complementary approaches can verify functional integrity:

• Reconstitution assays: Incorporation into proteoliposomes to measure proton translocation activity

• Circular dichroism (CD) spectroscopy: Verification of proper secondary structure, particularly the α-helical content expected for subunit c

• ATP synthesis coupling: When combined with other ATP synthase subunits, assessment of ATP production driven by artificially generated proton gradients

• Oligomerization analysis: Blue native PAGE or analytical ultracentrifugation to confirm formation of c-rings

• Proton binding capacity: Measurement of pH-dependent conformational changes using fluorescent probes

Each of these techniques provides different but complementary information about the structural and functional integrity of the purified protein .

What methods can identify interactions between atpH and other ATP synthase subunits?

Research on ATP synthase assembly requires sophisticated techniques to capture transient and stable interactions:

• Co-immunoprecipitation (Co-IP): Using antibodies against tagged atpH to pull down interaction partners

• Surface plasmon resonance (SPR): Quantitative measurement of binding kinetics between immobilized atpH and other subunits

• Förster resonance energy transfer (FRET): Detection of nanometer-scale proximity between fluorescently labeled subunits

• Crosslinking coupled with mass spectrometry: Identification of specific residues involved in subunit interfaces

• Yeast two-hybrid or bacterial two-hybrid systems: In vivo detection of protein-protein interactions

These approaches have revealed that subunit c interacts primarily with subunits a and b of the F0 sector, as well as with the γ and ε subunits of F1, forming the rotary mechanism that couples proton movement to ATP synthesis .

How is the expression of atpH regulated in chloroplasts?

The expression of atpH in chloroplasts involves complex regulatory mechanisms:

• Transcriptional control: Light-responsive elements in promoter regions modulate transcription rates

• Post-transcriptional regulation: RNA processing and stability affected by RNA-binding proteins

• Translational regulation: Within the translational feedback loop of ATP synthase assembly, production of nucleus-encoded subunit γ is required for sustained translation of chloroplast-encoded subunit β, which then stimulates expression of chloroplast-encoded subunit α

• Stoichiometric control: Translational downregulation occurs when subunits are not assembled, mediated through the 5'UTRs of their mRNAs

• Assembly-dependent regulation: Subunit γ releases negative feedback exerted by α/β assembly intermediates on translation of subunit β

This multilevel regulation ensures the precise 3:3:1:1:1 stoichiometry of the F1 sector (α:β:γ:δ:ε) required for functional assembly of the chloroplast enzyme .

What factors influence proper assembly of atpH into functional ATP synthase complexes?

Assembly of atpH into functional ATP synthase involves several critical factors:

• Sequential assembly pathway: The c-ring forms early in assembly, followed by attachment of other F0 components and then the F1 sector

• Chaperone assistance: Specific assembly factors aid in proper folding and integration

• Membrane composition: Specific lipid requirements, particularly cardiolipin in mitochondria or galactolipids in chloroplasts

• Environmental conditions: pH and ionic strength affect the oligomerization of subunit c

• Post-translational modifications: Potential regulatory modifications affecting assembly kinetics

Studies with ATP synthase mutants of Chlamydomonas reinhardtii have shown that defects in the expression of any constituent subunit lead to a pleiotropic loss in most polypeptides from both CF0 or CF1, indicating that assembly of the chloroplast ATP synthase is a highly concerted process .

How does pH affect the conformation and function of atpH in experimental settings?

Recent research has revealed significant pH-dependent structural dynamics of ATP synthase:

• At acidic pH (below neutral), ATP synthase exhibits four distinct conformations, three of which represent different stages in the enzyme's reaction cycle

• Two unique conformational states were recently identified under acidic conditions that had not been previously characterized

• These conformational changes directly affect the c-ring rotation and coupling efficiency

• Hypoxic conditions, which often lead to acidification, trigger these conformational changes with implications for enzyme function

• Mutations affecting pH sensitivity can significantly alter enzyme kinetics and efficiency

These findings are particularly relevant for understanding ATP synthase function in disease states characterized by altered tissue pH, such as cancer and cardiac ischemia. Studying atpH under various pH conditions provides insights into how the enzyme adapts to changing physiological environments .

What are the known inhibitors of ATP synthase that specifically target the c subunit?

Several inhibitors target the c subunit with varying specificity and mechanisms:

These inhibitors serve as valuable research tools for studying ATP synthase function and as potential leads for therapeutic applications, especially in contexts where inhibition of ATP synthase activity is desirable, such as in cancer treatment or antimicrobial therapy .

What roles does ATP synthase dysfunction play in human diseases?

ATP synthase dysfunction has been implicated in numerous human diseases:

• Neurodegenerative conditions:

  • In Alzheimer's disease, low expression of ATP synthase β subunit and cytosolic accumulation of α subunit are observed

  • Leigh syndrome can result from mutations in ATP synthase components

• Metabolic disorders:

  • Subunit c accumulation is characteristic of Batten's disease (neuronal ceroid lipofuscinoses)

  • Mitochondrial myopathies often involve ATP synthase deficiencies

• Cardiovascular conditions:

  • The F6 subunit of ATP synthase circulating in blood affects blood pressure regulation

  • Cardiac ischemia impacts ATP synthase function through pH-dependent mechanisms

• Cancer biology:

  • ATP synthase on the surface of endothelial cells plays a role in angiogenesis required for tumor growth

  • The β subunit serves as a target for innate antitumor cytotoxicity

These diverse pathological associations highlight the central importance of ATP synthase in cellular energy metabolism and its potential as a therapeutic target .

How can studies of recombinant atpH contribute to drug discovery?

Research using recombinant atpH can advance drug discovery through several approaches:

• Structure-based drug design targeting the c-ring:

  • Crystallographic or cryo-EM structures of recombinant atpH in various states provide templates for in silico screening

  • Conformational studies at different pH levels reveal potential druggable pockets

• High-throughput screening platforms:

  • Reconstituted proteoliposomes containing recombinant atpH can be used to screen compound libraries

  • FRET-based assays monitoring c-ring rotation offer sensitive detection of inhibitor activity

• Selective targeting strategies:

  • Exploiting structural differences between chloroplastic, bacterial, and human ATP synthase c subunits for antimicrobial specificity

  • Current examples include bedaquiline (Sirturo), an FDA-approved drug targeting mycobacterial ATP synthase for tuberculosis treatment

• Natural product research:

  • Identifying polyphenols and peptides that bind to distinctive sites at the interface of ATP synthase subunits

  • Determining structure-activity relationships through systematic modification of lead compounds

These approaches hold promise for developing new therapeutics for infections, cancer, and mitochondrial diseases .

What emerging techniques might advance our understanding of atpH function?

Several cutting-edge technologies show promise for deeper insights into atpH:

• Single-molecule biophysics:

  • Real-time visualization of c-ring rotation using high-speed atomic force microscopy

  • Optical tweezers to measure torque generation during proton translocation

• Synthetic biology approaches:

  • Engineering ATP synthase with modified c-rings to alter H+/ATP ratios

  • Creating minimal synthetic ATP synthase systems to define essential components

• Advanced structural methods:

  • Time-resolved cryo-EM to capture transient conformational states

  • Integrative structural biology combining multiple data types (cryo-EM, X-ray, NMR, mass spectrometry)

• Systems biology:

  • Multi-omics approaches to map ATP synthase interaction networks

  • Computational modeling of proton flow through the c-ring at atomic resolution

These emerging techniques will likely provide unprecedented insights into the molecular mechanics of ATP synthesis and the specific role of atpH .

What are the current challenges in atpH research and potential solutions?

Researchers face several persistent challenges when working with atpH:

• Membrane protein expression and stability:

  • Challenge: Low yields and protein aggregation during recombinant expression

  • Solution: Development of specialized expression hosts and membrane mimetics (nanodiscs, SMALPs)

• Functional reconstitution:

  • Challenge: Maintaining activity during purification and reconstitution

  • Solution: Novel detergent systems and co-expression with other ATP synthase components

• Structural heterogeneity:

  • Challenge: Multiple conformational states complicate structural analysis

  • Solution: New computational approaches for sorting heterogeneous structural datasets

• Translation to therapeutic applications:

  • Challenge: Selective targeting of pathogen ATP synthase without affecting human homologs

  • Solution: Detailed comparative structural analysis to identify unique features

• Integration with cellular context:

  • Challenge: Understanding how atpH function is influenced by cellular environment

  • Solution: In-cell structural biology techniques and advanced imaging approaches

Addressing these challenges will require interdisciplinary approaches combining structural biology, biochemistry, biophysics, and computational methods .