Recombinant Nephroselmis olivacea ATP synthase subunit c, chloroplastic (atpH)

What is the role of ATP synthase subunit c in chloroplastic energy metabolism?

The subunit c of chloroplastic ATP synthase forms a critical component of the multimeric ATP synthase complex that produces adenosine triphosphate (ATP) required for photosynthetic metabolism. It forms an oligomeric ring (cₙ ring) embedded in the thylakoid membrane, where its rotation is mechanically coupled to ATP synthesis. This rotation is driven by proton translocation across the membrane along an electrochemical gradient, forming the basis of the chemiosmotic theory of energy conversion . The c-subunit ring functions as the rotor component of this molecular motor, with each c-subunit containing an essential proton-binding site that facilitates proton movement through the membrane.

Why is ATP synthase sometimes incorrectly referred to as ATPase in literature?

The terminology confusion between ATP synthase and ATPase stems from historical experimental contexts. Early research focused on the enzyme's ability to hydrolyze ATP (ATPase activity) before its primary physiological role in synthesizing ATP was fully established. In the 1960s, researchers like Kagawa and Racker isolated mitochondrial fractions with ATPase activity while attempting to understand ATP synthesis . The complete ATP synthase structure only emerged in the 1990s, confirming its primary function as a synthase.

What is the genetic organization of the atpH gene in chloroplast genomes?

The atpH gene, encoding ATP synthase subunit c, is typically located within a gene cluster in the chloroplast genome. In Chlamydomonas reinhardtii, for example, the atpH gene is part of the atpA gene cluster, which includes atpA, psbI, cemA, and atpH genes . The expression of atpH involves specific regulatory elements:

• Transcription initiation: The atpH gene possesses its own promoter with a distinct 5' end preceded by a palindromic TATAAT(AT) consensus sequence positioned at approximately -13 relative to the mature 5' end .

• Regulatory elements: A- and T-rich sequences typically surround or lie immediately upstream of the atpH 5' end, although these may not always match the consensus sequences found in other chloroplast genes .

• Experimental verification: The promoter activity of atpH has been confirmed through reporter gene fusion experiments, where upstream regions were fused to reporter genes like the E. coli uidA gene (encoding β-glucuronidase/GUS) and successfully expressed in chloroplasts after transformation .

What strategies have proven successful for recombinant expression of chloroplastic ATP synthase subunit c?

Recombinant expression of chloroplastic ATP synthase subunit c presents significant challenges due to its highly hydrophobic nature and membrane-embedded location. Based on successful strategies with spinach chloroplast ATP synthase subunit c, a methodological approach can be developed for Nephroselmis olivacea:

• Expression system selection: BL21 derivative Escherichia coli cells have proven effective for expressing eukaryotic membrane proteins like ATP synthase subunit c .

• Gene optimization: Codon optimization of the atpH gene for E. coli expression is critical to overcome codon bias issues and enhance expression levels .

• Fusion protein approach: Expression as a soluble fusion protein with maltose binding protein (MBP) significantly improves solubility and yield. The general construct design includes:

  • N-terminal MBP domain

  • Suitable linker region

  • Protease cleavage site

  • ATP synthase subunit c sequence

• Expression conditions: Optimization of parameters including:

  • Induction temperature (typically lowered to 16-25°C)

  • IPTG concentration

  • Induction duration

  • Media composition

This approach has successfully produced significant quantities of properly folded chloroplastic ATP synthase subunit c with the correct α-helical secondary structure .

What purification methods are most effective for isolating recombinant ATP synthase subunit c?

Purification of recombinant ATP synthase subunit c requires a strategic approach addressing its hydrophobic nature and need for structural integrity:

• Initial affinity purification: For MBP-fusion constructs, amylose resin chromatography effectively captures the fusion protein:

  • Binding: Cell lysate is applied to amylose resin in appropriate buffer conditions

  • Washing: Multiple wash steps remove contaminants

  • Elution: Controlled elution with maltose yields purified fusion protein

• Protease cleavage: The fusion protein undergoes controlled proteolytic cleavage to separate the MBP tag from ATP synthase subunit c:

  • Optimized protease:substrate ratio

  • Controlled temperature and duration

  • Buffer conditions preserving subunit c structure

• Reversed-phase chromatography: Final purification of cleaved ATP synthase subunit c is achieved through reversed-phase column chromatography:

  • Column selection: C4 or C8 columns typically work well for hydrophobic membrane proteins

  • Mobile phase: Gradient of organic solvent (acetonitrile) with trifluoroacetic acid

  • Fraction collection and analysis

• Verification of structural integrity: Circular dichroism spectroscopy confirms the correct α-helical secondary structure of the purified subunit c .

How does the c-ring stoichiometry vary between different organisms and what factors contribute to this variation?

The c-ring stoichiometry (the number of c-subunits per oligomeric ring) varies across different organisms and has significant implications for the bioenergetics of ATP synthesis:

• Stoichiometric variation: The number of c-subunits (n) per oligomeric ring (cₙ) varies between organisms, typically ranging from 8 to 15 subunits .

• Bioenergetic implications: This variation directly affects the ratio of protons translocated to ATP synthesized, since each complete rotation of the c-ring (requiring protonation/deprotonation of each c-subunit) produces a fixed number of ATP molecules .

• Contributing factors: While not fully understood, several factors potentially influence c-ring stoichiometry:

  • Evolutionary adaptation to specific metabolic requirements

  • Membrane lipid composition and thickness

  • Environmental pH conditions

  • Energetic efficiency requirements of the organism

• Methodological approaches to study stoichiometry:

  • Cryo-electron microscopy of isolated c-rings

  • Mass spectrometry of intact c-rings

  • Cross-linking studies combined with electrophoretic analysis

  • Atomic force microscopy of membrane-embedded ATP synthase complexes

Understanding these variations in Nephroselmis olivacea would require isolation and structural characterization of its native or recombinantly expressed c-ring.

How does pH affect the function and conformation of ATP synthase subunit c?

The function of ATP synthase subunit c is intrinsically pH-sensitive due to its role in proton translocation:

• pH-dependent conformational changes: Single-molecule spectroscopic studies reveal that protonation/deprotonation events trigger specific conformational changes that generate torque for ATP synthesis .

• Mechanistic insights:

  • Subunit-a interactions: The interface between subunit-a and the c-ring is particularly sensitive to pH changes

  • Critical residues: Specific amino acids with pKa values near physiological pH serve as proton-binding sites

  • Sub-step mechanics: ATP synthase exhibits pH-dependent sub-steps during rotation, with distinct transitions observed at different pH values

• Experimental data on pH sensitivity:

  • Wild-type ATP synthase shows pH-dependent transition dwell (TD) formation with pKa₁ of approximately 6.5 and pKa₂ of approximately 7.7

  • Mutations in key residues shift these pKa values, altering the pH-dependence profile

  • Maximum TD formation occurs at optimal pH ranges specific to each organism's physiological environment

These pH-dependent properties would be critical to consider when designing experimental conditions for studying Nephroselmis olivacea ATP synthase subunit c.

What is the relationship between ATP synthase subunit c and calcium-dependent membrane permeabilization?

Recent research has uncovered unexpected roles for ATP synthase subunit c beyond its canonical function in ATP synthesis:

• Amyloidogenic properties: ATP synthase subunit c has been identified as an amyloidogenic peptide capable of spontaneously folding into β-sheets and self-assembling into fibrils and oligomers in a Ca²⁺-dependent manner .

• Membrane permeabilization: Under certain conditions, subunit c may participate in inner mitochondrial membrane (IMM) permeabilization through calcium-induced permeability transition .

• Structural transitions:

  • Native state: α-helical hairpin organized in oligomeric rings spanning the lipid bilayer

  • Alternative folding: Under specific conditions, particularly elevated calcium, subunit c can adopt β-sheet conformations associated with amyloid formation

  • Oligomerization: Formation of non-native oligomeric structures that may create membrane pores

• Experimental approaches:

  • Fluorescence spectroscopy for conformational analysis

  • Atomic force microscopy to visualize oligomer/fibril formation

  • Black lipid membrane methods to assess membrane permeabilization properties

While these studies focused on mitochondrial ATP synthase subunit c, the structural similarities with chloroplastic subunit c suggest potential parallel mechanisms that could be explored in Nephroselmis olivacea.

How can site-directed mutagenesis of ATP synthase subunit c inform our understanding of proton translocation mechanisms?

Site-directed mutagenesis of ATP synthase subunit c provides powerful insights into the molecular mechanisms of proton translocation and ATP synthesis:

• Identification of critical residues: Mutations targeting specific amino acids can reveal those essential for:

  • Proton binding and release

  • Conformational changes during rotation

  • Subunit-subunit interactions

  • Structural stability of the c-ring

• pH sensitivity alterations: Mutations in key residues can shift the pH-dependence profile:

  • aN214L mutation increases pKa₁ by 0.9 units and pKa₂ by 0.7 units

  • aQ252L and aE219L mutations increase pKa₁ by 0.6 and 0.5 units respectively, while decreasing pKa₂

• Functional consequences: Specific mutations result in measurable changes:

  • Altered torque generation

  • Changed efficiency of ATP synthesis

  • Modified proton:ATP stoichiometry

  • Shifts in optimal pH range for function

• Experimental design considerations for Nephroselmis olivacea:

  • Identify conserved residues through sequence alignment

  • Target residues predicted to be involved in proton binding/release

  • Create a library of mutants with substitutions affecting charge, hydrophobicity, or steric properties

  • Express and characterize mutants using both in vitro and in vivo assays

What are the latest techniques for studying the structure-function relationship of ATP synthase subunit c?

Contemporary research employs sophisticated techniques to elucidate the structure-function relationship of ATP synthase subunit c:

• Cryo-electron microscopy (cryo-EM):

  • Near-atomic resolution structures of entire ATP synthase complexes

  • Visualization of different conformational states during the catalytic cycle

  • Integration of subunit c within the membrane environment

• Single-molecule spectroscopy:

  • FRET (Förster Resonance Energy Transfer) analysis of conformational changes

  • Direct observation of rotational steps during ATP synthesis

  • pH-dependent sub-step resolution

• Molecular dynamics simulations:

  • Atomistic modeling of proton movement through the c-ring

  • Simulation of conformational changes during rotation

  • Prediction of effects of mutations on structure and function

• Native mass spectrometry:

  • Determination of intact c-ring stoichiometry

  • Analysis of subunit interactions and stability

  • Detection of post-translational modifications

• In situ techniques:

  • Single-particle tracking in live cells

  • Super-resolution microscopy of ATP synthase distribution

  • Correlative light and electron microscopy

Application of these techniques to Nephroselmis olivacea ATP synthase subunit c would provide valuable comparative data to understand evolutionary conservation and specialization of this critical protein across photosynthetic organisms.

What challenges exist in studying the assembly of the c-ring structure and its integration into the ATP synthase complex?

Investigating the assembly of ATP synthase subunit c into functional c-rings and their integration into the complete ATP synthase complex presents several methodological challenges:

• Assembly pathway elucidation:

  • Temporal sequence of c-subunit oligomerization

  • Role of assembly factors/chaperones

  • Coordination with other ATP synthase subunit assembly

• Technical challenges:

  • Capturing assembly intermediates due to their transient nature

  • Maintaining native membrane environment during isolation

  • Distinguishing between functional and artifact oligomeric states

• Integration with other components:

  • Interaction interfaces between c-ring and a-subunit

  • Coupling mechanisms between F₁ and F₀ sectors

  • Role of peripheral stalk in stabilizing the assembled complex

• Methodological approaches:

  • Pulse-chase experiments with tagged subunits

  • Conditional expression systems

  • In vitro reconstitution from purified components

  • Time-resolved structural analysis during assembly

For Nephroselmis olivacea specifically, the chloroplastic environment introduces additional complexity due to the thylakoid membrane's unique lipid composition and the coordination of nuclear and chloroplast gene expression for the different ATP synthase subunits.

How does the sequence and structure of ATP synthase subunit c vary across different photosynthetic organisms?

The ATP synthase subunit c shows interesting patterns of conservation and variation across photosynthetic organisms:

• Sequence conservation:

  • Highly conserved proton-binding site (typically containing a critical glutamate or aspartate residue)

  • Conserved glycine-rich motifs facilitating helix-helix packing

  • Variable regions often correspond to lipid-facing surfaces

• Structural variations:

  • Length differences in transmembrane helices

  • Variable stoichiometry of c-rings (8-15 subunits)

  • Species-specific adaptations in surface residues

• Evolutionary implications:

  • Conservation of core functional elements across diverse photosynthetic lineages

  • Adaptations potentially reflecting different environmental pressures

  • Co-evolution with interacting ATP synthase subunits

• Methodological approach for comparative analysis:

  • Multiple sequence alignment of subunit c from diverse photosynthetic organisms

  • Phylogenetic analysis to trace evolutionary relationships

  • Structural modeling to predict functional consequences of variations

  • Experimental validation of predicted structural differences

Nephroselmis olivacea, as a prasinophyte green alga, occupies an interesting evolutionary position that could provide insights into the early evolution of chloroplastic ATP synthase in the green lineage.

What experimental approaches can determine whether recombinant Nephroselmis olivacea ATP synthase subunit c can functionally substitute for homologs from other species?

Testing the functional interchangeability of ATP synthase subunit c across species requires specialized approaches:

• Complementation studies:

  • Gene knockout/knockdown of native subunit c

  • Expression of Nephroselmis olivacea subunit c in heterologous systems

  • Assessment of ATP synthesis restoration

  • Measurement of growth and photosynthetic efficiency

• Chimeric c-ring formation:

  • Co-expression of tagged native and Nephroselmis subunit c

  • Analysis of mixed c-ring formation

  • Functional characterization of hybrid complexes

  • Stability assessment of chimeric rings

• In vitro reconstitution:

  • Purification of recombinant Nephroselmis olivacea subunit c

  • Combination with ATP synthase components from other species

  • Measurement of ATP synthesis/hydrolysis activities

  • Structural verification of assembled complexes

• Specific parameters to evaluate:

  • ATP synthesis rates

  • Proton translocation efficiency

  • c-ring stability

  • pH dependence profile

  • Sensitivity to known inhibitors

Such studies would provide valuable insights into the structural determinants of species-specific interactions within the ATP synthase complex.