Recombinant Brachypodium distachyon ATP synthase subunit c, chloroplastic (atpH)

What is the function of ATP synthase subunit c in Brachypodium distachyon chloroplasts?

ATP synthase subunit c forms the c-ring structure in the FO portion of the chloroplastic ATP synthase complex. This ring functions as a rotary motor driven by proton flow across the thylakoid membrane, ultimately enabling ATP synthesis in the F1 portion. In B. distachyon chloroplasts, as in other plants, this protein plays a crucial role in photosynthetic energy conversion.

For experimental determination of c-subunit function in recombinant systems, researchers typically employ:

• Site-directed mutagenesis of conserved proton-binding residues

• Reconstitution of purified protein into liposomes for functional assays

• Measurement of proton translocation using pH-sensitive fluorescent dyes

• Analysis of ATP synthesis coupling to proton gradients

The precise coupling ratio between proton translocation and ATP synthesis depends on the c-ring stoichiometry, which affects the bioenergetic efficiency of the entire complex.

What expression systems are most effective for producing recombinant B. distachyon atpH?

Production of functional recombinant ATP synthase subunit c poses significant challenges due to its hydrophobic nature. Several expression systems have been optimized for membrane proteins like atpH:

• Bacterial expression (E. coli):

  • Use of C41/C43 strains specifically developed for membrane proteins

  • Expression as fusion proteins with solubility tags (MBP, SUMO)

  • Inclusion body production followed by refolding protocols

• Yeast expression (P. pastoris):

  • Methanol-inducible expression under AOX1 promoter

  • Secretion signals for improved processing

  • Scale-up capability in bioreactors

• Plant-based expression:

  • Agrobacterium-mediated transformation of B. distachyon

  • Transient expression in Nicotiana benthamiana

  • Chloroplast transformation for organelle-targeted expression

For B. distachyon specifically, transformation protocols similar to those used for other transgenic studies (such as IRI gene knockdowns) can be adapted, involving selection on hygromycin-containing media following Agrobacterium-mediated transformation .

What techniques are most effective for purifying recombinant atpH?

Purification of recombinant ATP synthase subunit c requires specialized approaches for membrane proteins:

• Membrane isolation and solubilization:

  • Differential centrifugation to isolate membrane fractions

  • Selective solubilization with mild detergents (DDM, LMNG)

  • Detergent screening to maintain native oligomeric state

• Chromatographic separation:

  • Immobilized metal affinity chromatography (IMAC) using engineered His-tags

  • Size exclusion chromatography to separate oligomeric states

  • Ion exchange chromatography for final polishing

• Functional reconstitution:

  • Incorporation into nanodiscs for structural studies

  • Proteoliposome reconstitution for functional assays

  • Detergent exchange for crystallization trials

How does pH regulation affect the function of chloroplastic ATP synthase?

The pH gradient across the thylakoid membrane provides the driving force for ATP synthesis. Research on plant pH regulation provides important context for understanding this process:

Advanced pH measurement methods, such as membrane-anchored ratiometric pH sensors allowing non-invasive measurement of pH gradients , could be adapted to study thylakoid pH regulation in B. distachyon chloroplasts.

What structural analysis techniques are most suitable for studying the c-ring of recombinant B. distachyon ATP synthase?

High-resolution structural determination of membrane protein complexes like ATP synthase requires specialized approaches:

• X-ray crystallography:

  • In meso crystallization using lipidic cubic phases, which proved successful for spinach chloroplast c-ring structure determination at 2.3 Å resolution

  • Screening of detergent and lipid compositions to optimize crystal packing

  • Synchrotron radiation with microfocus beamlines for small crystals

• Cryo-electron microscopy:

  • Single-particle analysis of detergent-solubilized complexes

  • Focused classification and refinement strategies for flexible regions

  • Direct electron detectors for improved signal-to-noise ratio

• Integrative approaches:

  • Mass spectrometry to determine subunit stoichiometry

  • Molecular dynamics simulations to model proton translocation

  • Cross-linking mass spectrometry to map subunit interactions

Electron density features inside the c-ring, as observed in the spinach chloroplast structure , may represent isoprenoid quinones (such as plastoquinone in chloroplasts) that could serve as universal cofactors of ATP synthases, stabilizing the c-ring structure .

How can researchers effectively investigate the c-ring stoichiometry in B. distachyon ATP synthase?

The number of c subunits in the ring directly affects the H+/ATP ratio and energy conversion efficiency. Several complementary approaches can determine this crucial parameter:

• Direct structural methods:

  • X-ray crystallography or cryo-EM to directly visualize and count subunits

  • Atomic force microscopy of membrane-embedded complexes

  • Native mass spectrometry of intact c-rings

• Biochemical approaches:

  • Cross-linking followed by SDS-PAGE analysis

  • Quantitative amino acid analysis

  • Chemical labeling of essential residues

• Functional methods:

  • Measurement of H+/ATP ratio in reconstituted systems

  • Thermodynamic analysis of ATP synthesis under defined PMF conditions

The spinach chloroplast c-ring contains 14 subunits , and this stoichiometry may be conserved in B. distachyon as a fellow plant species, though experimental verification is essential to confirm this prediction.

What methodologies can assess the impact of cold stress on B. distachyon ATP synthase function?

B. distachyon has been studied for its response to cold stress , and these stress conditions likely affect ATP synthase function through multiple mechanisms:

• Expression and regulatory studies:

  • Cold-inducible promoters like prOsMYB1R35 from rice can be used to study atpH expression regulation

  • Quantitative proteomics to measure ATP synthase subunit abundance changes

  • Transcriptional analysis of chloroplastic genes under cold conditions

• Functional analyses:

  • Oxygen evolution and chlorophyll fluorescence measurements

  • ATP synthesis rates in isolated chloroplasts at different temperatures

  • Proton gradient formation using pH-sensitive fluorescent probes

• Structural studies:

  • Thermal stability assays to determine effects on complex integrity

  • Hydrogen-deuterium exchange mass spectrometry to identify regions with altered dynamics

  • Comparative analysis with cold-adapted species

The cold-inducible promoter system used in B. distachyon for studying ice recrystallization inhibition genes provides a methodological framework that could be adapted for investigating cold stress effects on ATP synthase.

How do mutations in atpH affect proton translocation and ATP synthesis?

Mutagenesis studies provide crucial insights into structure-function relationships in ATP synthase. For B. distachyon atpH, several approaches are particularly informative:

• Site-directed mutagenesis targets:

  • Conserved proton-binding residues (typically Asp or Glu in TM2)

  • Residues at subunit interfaces critical for c-ring assembly

  • Residues involved in interactions with other ATP synthase components

• Expression and analysis systems:

  • Complementation of ATP synthase-deficient E. coli strains

  • Chloroplast transformation for homologous expression

  • In vitro reconstitution systems with defined components

• Functional readouts:

  • ATP synthesis rates under defined conditions

  • Proton leakage measurements using pH indicators

  • Rotational analysis using single-molecule techniques

Transformation protocols established for B. distachyon, such as those using artificial miRNA driven by specific promoters , provide effective methods for introducing mutations into the endogenous atpH gene.

What are the optimal methods for investigating the role of lipids in c-ring function?

The lipid environment significantly affects membrane protein function, including ATP synthase:

• Lipid analysis techniques:

  • Lipidomics of purified ATP synthase complexes

  • Native mass spectrometry to identify tightly bound lipids

  • Molecular dynamics simulations to predict lipid binding sites

• Functional reconstitution approaches:

  • Systematic variation of lipid composition in proteoliposomes

  • Native nanodiscs with defined lipid environments

  • Reconstitution into giant unilamellar vesicles for single-complex studies

• Specific lipid interactions:

  • Photocrosslinking with lipid analogs

  • Fluorescence quenching assays for lipid binding

  • EPR spectroscopy with spin-labeled lipids

The electron densities observed inside ATP synthase c-rings from various species may represent bound isoprenoid molecules like plastoquinone in chloroplasts, suggesting a universal role for these molecules in stabilizing the c-ring structure and potentially preventing ion leakage .

How can researchers study the assembly process of the c-ring in B. distachyon chloroplasts?

Assembly of the c-ring is a complex process essential for ATP synthase function:

• In vivo assembly studies:

  • Pulse-chase experiments with radiolabeled amino acids

  • Inducible expression systems to synchronize assembly

  • Isolation of assembly intermediates

• Identification of assembly factors:

  • Co-immunoprecipitation of interacting proteins

  • Proximity labeling approaches (BioID, APEX)

  • Genetic screens for assembly-defective mutants

• Visualization of assembly:

  • Fluorescence microscopy of tagged subunits

  • Time-resolved cryo-EM to capture assembly intermediates

  • Single-molecule FRET to monitor conformational changes

The striking feature of circular-like electron densities in the hydrophobic part of the internal pore of ATP synthase c-rings suggests these structures may play a role in stabilizing the assembled complex across species from archaea and bacteria to eukaryotes.

What techniques can evaluate the proton pathway through the B. distachyon ATP synthase c-ring?

Proton translocation through the c-ring drives ATP synthesis, making this pathway crucial to understand:

• Structural approaches:

  • X-ray crystallography at different pH values

  • Neutron diffraction to directly visualize proton positions

  • Molecular dynamics simulations with specialized force fields

• Spectroscopic methods:

  • FTIR difference spectroscopy to detect protonation changes

  • Solid-state NMR of labeled residues in the proton path

  • Time-resolved fluorescence with pH-sensitive probes

• Electrophysiological techniques:

  • Patch-clamp of reconstituted c-rings

  • Solid-supported membrane electrophysiology

  • Ion conductance measurements in artificial bilayers

The unusual features observed in ATP synthase c-rings, including the large distance between polar/apolar interfaces inside the ring , may play critical roles in controlling proton translocation and preventing leakage.