Recombinant Hordeum vulgare ATP synthase subunit c, chloroplastic (atpH)

Basic Research Questions

• What is the structure and function of ATP synthase subunit c in chloroplasts?

  ATP synthase subunit c (atpH) in Hordeum vulgare is a small hydrophobic protein (81 amino acids) that forms part of the membrane-embedded F0 sector of the chloroplast ATP synthase complex . Multiple copies of subunit c assemble into a ring structure (c-ring) within the thylakoid membrane, which plays a crucial role in proton translocation and the rotary mechanism of ATP synthesis.

  Structurally, the protein contains two transmembrane α-helices connected by a polar loop region. The high hydrophobicity of the protein is evident from its amino acid sequence: MNPLIAAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV . This hydrophobicity facilitates its integration into the lipid bilayer of the thylakoid membrane.

  Functionally, the c-ring rotates as protons flow through the F0 sector, driven by the proton motive force (pmf) generated during photosynthetic electron transport . This rotation is mechanically coupled to conformational changes in the F1 sector, where ATP synthesis occurs. Unlike its counterparts in mitochondria or bacteria, the chloroplast ATP synthase features unique regulatory mechanisms, particularly redox regulation mediated by thioredoxin, which activates the enzyme in the light and inactivates it in the dark .

• How is recombinant Hordeum vulgare ATP synthase subunit c expressed and purified for research applications?

  Recombinant expression of Hordeum vulgare ATP synthase subunit c typically employs E. coli expression systems due to their high yield and relative simplicity . The gene encoding the full-length protein (amino acids 1-81) is cloned into an expression vector that introduces an N-terminal His-tag to facilitate purification.

  A standardized protocol for expression and purification includes:

  Expression:

  • Transform the expression construct into an E. coli strain (commonly BL21(DE3))

  • Culture in LB medium with appropriate antibiotics at 37°C until mid-log phase

  • Induce protein expression with IPTG (typically 0.5-1 mM)

  • Continue cultivation for 4-6 hours or overnight at reduced temperature (16-25°C)

  Purification:

  • Harvest cells by centrifugation and disrupt using sonication or mechanical methods

  • Solubilize membrane fractions using mild detergents (e.g., DDM, LDAO)

  • Perform affinity chromatography using Ni-NTA resin to capture the His-tagged protein

  • Wash extensively to remove non-specifically bound proteins

  • Elute with an imidazole gradient

  • Further purify by size-exclusion chromatography if necessary

  The purified protein is typically obtained as a lyophilized powder and should be stored at -20°C/-80°C . For experimental use, reconstitution in Tris/PBS-based buffer with 6% trehalose at pH 8.0 is recommended, with the addition of 5-50% glycerol for long-term storage .

• How does chloroplast ATP synthase differ from mitochondrial and bacterial ATP synthases?

  Chloroplast ATP synthase shares the basic F-type architecture with mitochondrial and bacterial ATP synthases but exhibits several distinctive features relevant to research on the atpH subunit:

  Redox regulation: The most prominent distinguishing feature of chloroplast ATP synthase is its redox regulation system . The enzyme is activated in the light and inactivated in the dark through a thioredoxin-mediated mechanism. This regulation involves two cysteines located on the γ subunit that form a disulfide bridge in the dark, inhibiting rotation and ATP synthesis/hydrolysis .

  Adaptation to light-dark cycles: Recent research has revealed that this redox regulation affects more than just ATP hydrolysis. Studies in Arabidopsis comparing wild-type plants with mutants expressing redox-insensitive ATP synthase found that plants with normal redox regulation lost photosynthetic capacity more rapidly in darkness . This suggests that down-regulation of ATP synthase in the dark leads to dissipation of proton motive force, affecting protein transport across the thylakoid membrane and maintenance of photosynthetic complexes .

  Structural adaptations: The c-ring of chloroplast ATP synthase typically contains a different number of c subunits compared to mitochondrial or bacterial counterparts, reflecting adaptations to the specific energy requirements of photosynthesis.

  Proton source: While mitochondrial ATP synthase utilizes the proton gradient generated by the respiratory chain, chloroplast ATP synthase uses the proton gradient established by photosynthetic electron transport.

  Understanding these differences is crucial when designing experiments with recombinant chloroplast ATP synthase components, as the functional context differs significantly from other ATP synthases.

• What experimental approaches can be used to study c-ring assembly using recombinant subunit c?

  Several experimental approaches can be employed to study c-ring assembly using recombinant Hordeum vulgare ATP synthase subunit c:

  In vitro reconstitution:

  • Express and purify recombinant subunit c with appropriate tags

  • Solubilize in mild detergents or lipid nanodiscs

  • Allow spontaneous self-assembly under controlled conditions

  • Analyze assembled structures by electron microscopy or native PAGE

  Fluorescence-based assembly monitoring:

  • Introduce strategically positioned cysteine residues for fluorophore labeling

  • Monitor FRET between labeled subunits during assembly

  • Track assembly kinetics under various conditions (pH, lipid composition, salt)

  Cross-linking approaches:

  • Use chemical cross-linkers of various lengths to capture assembly intermediates

  • Apply mass spectrometry to identify interaction interfaces

  • Perform time-course experiments to follow assembly progression

  Single-molecule visualization:

  • Employ high-speed atomic force microscopy to directly observe assembly

  • Track the addition of individual subunits to growing c-rings

  • Analyze assembly pathways and potential intermediate states

  Co-expression strategies:

  • Co-express subunit c with other F0 components in E. coli

  • Isolate intact subcomplexes

  • Analyze composition and stoichiometry by mass spectrometry

  These approaches provide complementary information about the assembly process, allowing researchers to develop a comprehensive understanding of how individual subunit c proteins organize into functional c-rings.

Advanced Research Questions

• How does redox regulation affect the activity of chloroplast ATP synthase, and what implications does this have for studies using recombinant subunit c?

  Redox regulation is a critical control mechanism for chloroplast ATP synthase activity, with significant implications for research utilizing recombinant components like subunit c.

  Mechanism of redox regulation:
  The chloroplast ATP synthase is regulated by the thioredoxin system, which is directly coupled to photosynthetic electron transport . Two cysteine residues located on the γ subunit form a disulfide bridge in the dark, inhibiting rotation and ATP synthesis. In the light, photosynthetically reduced thioredoxin reduces this disulfide bond, allowing rotation to proceed .

  Recent research has challenged traditional views about this regulation. Studies comparing wild-type Arabidopsis with mutants expressing redox-insensitive ATP synthase revealed that plants with normal redox regulation lost photosynthetic capacity rapidly in darkness, while plants with constitutively active ATP synthase maintained photosynthetic activity longer . This suggests that down-regulation in the dark leads to dissipation of the proton motive force (pmf), inhibiting protein transport across the thylakoid membrane and resulting in the selective loss of photosynthetic complexes .

  Experimental implications:

  Experimental Aspect	Methodological Considerations
  Reconstitution studies	Must include γ subunit and maintain appropriate redox environment
  Functional assays	Should be conducted under defined redox conditions
  Protein-protein interactions	Interactions between c-ring and γ subunit may vary with redox state
  Structural analysis	Conformational changes may occur depending on redox conditions
  Rotation measurements	Rotation dynamics will differ based on redox state of the complex

  When designing experiments with recombinant subunit c, researchers must consider whether to recreate the native redox regulation system or to work with a simplified system lacking this regulation. The choice depends on the specific research questions being addressed and the degree to which physiological relevance is required.

• What are the challenges and solutions for maintaining the native structure of recombinant ATP synthase subunit c during in vitro studies?

  Maintaining the native structure of recombinant ATP synthase subunit c presents several challenges due to its highly hydrophobic nature and its normal integration within a membrane environment. Here are the key challenges and methodological solutions:

  Challenges:

  1. Protein aggregation: The hydrophobic nature of subunit c makes it prone to aggregation in aqueous solutions.

  2. Misfolding: Without the membrane environment or chaperone assistance, recombinant subunit c may not fold into its native conformation.

  3. Stability issues: The isolated protein may be less stable than when integrated into the complete ATP synthase complex.

  4. Oligomerization: Subunit c naturally forms a multimeric ring structure; ensuring proper oligomerization in vitro can be difficult.

  Methodological solutions:

  1. Detergent selection:

     • Use mild, non-denaturing detergents (e.g., DDM, LDAO, Brij-35)

     • Perform detergent screening to identify optimal conditions

     • Consider novel detergents like maltose-neopentyl glycol compounds

  2. Membrane mimetics:

     • Nanodiscs: Phospholipid bilayers encircled by membrane scaffold proteins

     • Liposomes: For functional reconstitution experiments

     • Bicelles: Disk-shaped lipid-detergent mixed micelles

     • Amphipols: Amphipathic polymers that stabilize membrane proteins

  3. Buffer optimization:

     • Include glycerol (5-50%) to prevent aggregation

     • Add stabilizing agents like trehalose (6%)

     • Maintain optimal pH (typically pH 8.0 for storage)

     • Use low ionic strength buffers for initial solubilization

  4. Structural validation:

     • Circular dichroism (CD) to confirm secondary structure content

     • Size-exclusion chromatography to assess oligomeric state

     • Limited proteolysis to evaluate folding quality

     • Functional assays to confirm activity when reconstituted

  By addressing these challenges with appropriate methodological solutions, researchers can maintain the native structure of recombinant ATP synthase subunit c and improve the reliability of their in vitro studies.

• How can researchers effectively incorporate recombinant ATP synthase subunit c into artificial membrane systems for functional studies?

  Incorporating recombinant ATP synthase subunit c into artificial membrane systems is crucial for functional studies but requires careful methodological consideration. Below are detailed approaches for effective reconstitution:

  Liposome reconstitution:

  1. Lipid selection and preparation:

     • Use a mixture of phospholipids that mimics the native thylakoid membrane composition

     • Typical mixtures include DOPC/DOPE/DOPG at ratios similar to chloroplast membranes

     • Dissolve purified lipids in chloroform and create a thin film by evaporation

     • Hydrate the lipid film with buffer containing detergent

  2. Reconstitution protocol:

     • Add purified recombinant subunit c at a protein-to-lipid ratio of 1:50 to 1:200 (w/w)

     • Remove detergent by dialysis, Bio-Beads adsorption, or gel filtration

     • For functional c-rings, co-reconstitute with other necessary F0 components

     • Control protein orientation by pH gradients during reconstitution

  3. Verification methods:

     • Freeze-fracture electron microscopy to visualize protein incorporation

     • Fluorescence recovery after photobleaching (FRAP) to assess mobility

     • Sucrose density gradient centrifugation to separate proteoliposomes

  Nanodiscs system:

  1. Assembly protocol:

     • Express and purify membrane scaffold protein (MSP)

     • Mix purified subunit c, MSP, and lipids in detergent solution (molar ratio ~1:2:120)

     • Remove detergent using Bio-Beads or dialysis

     • Purify assembled nanodiscs by size-exclusion chromatography

  Functional assessment strategies:

  1. Proton translocation assays:

     • Use pH-sensitive fluorescent dyes (e.g., ACMA, pyranine)

     • Create a pH gradient across the membrane

     • Monitor fluorescence changes indicative of proton movement

  2. Rotation assays for assembled c-rings:

     • Attach fluorescent probes or gold nanoparticles to the c-ring

     • Apply a proton motive force

     • Visualize rotation using fluorescence microscopy or dark-field microscopy

  Method	Advantages	Limitations	Best Applications
  Liposomes	Bilayer environment, variable size	Heterogeneous orientation	Bulk functional assays
  Nanodiscs	Controlled size, homogeneous	Small size limits complex formation	Structural studies, binding assays
  Planar bilayers	Electrical access to both sides	Technical complexity	Electrophysiology measurements
  GUVs (Giant Unilamellar Vesicles)	Visualization capability	Fragility	Single-molecule studies

  These methodologies provide researchers with multiple approaches to incorporate recombinant ATP synthase subunit c into artificial membrane systems, enabling detailed functional characterization of this essential component of the ATP synthase complex.

• What experimental approaches can be used to study the rotation dynamics of the c-ring in ATP synthase using recombinant subunit c?

  Studying the rotation dynamics of the ATP synthase c-ring requires sophisticated biophysical techniques that can detect nanoscale movements in real-time. Here are detailed methodological approaches utilizing recombinant subunit c:

  Single-molecule fluorescence techniques:

  1. Fluorescence Resonance Energy Transfer (FRET):

     • Introduce cysteine residues at strategic positions in recombinant subunit c for site-specific labeling

     • Label with donor-acceptor fluorophore pairs (e.g., Cy3-Cy5)

     • Reconstitute labeled subunits into functional c-rings

     • Measure distance changes between fluorophores during rotation

     • Analyze FRET efficiency changes to determine step size and rotation rates

  2. Total Internal Reflection Fluorescence (TIRF) microscopy:

     • Immobilize ATP synthase complexes containing fluorescently labeled c-rings on a glass surface

     • Visualize rotation through the movement of the attached fluorophore

     • Record long trajectories at frame rates of 30-100 fps

     • Analyze rotational velocity, step size, and dwell times

  High-speed AFM (HS-AFM):

  1. Sample preparation and imaging:

     • Reconstitute c-rings in supported lipid bilayers

     • Operate in tapping mode with soft cantilevers (spring constant ~0.1-0.2 N/m)

     • Achieve frame rates of 5-10 fps

     • Maintain minimal imaging forces (<100 pN)

  2. Data analysis:

     • Develop image processing algorithms to track rotational movement

     • Calculate angular velocity and step size

     • Correlate structural changes with functional states

  Gold nanoparticle labeling and dark-field microscopy:

  1. Nanoparticle attachment:

     • Engineer recombinant subunit c with accessible thiol groups or specific tags

     • Conjugate gold nanoparticles (40-100 nm) to modified subunits

     • Incorporate labeled subunits into functional c-rings

  2. Observation system:

     • Use dark-field microscopy to detect scattered light from gold particles

     • Record at high frame rates (1000+ fps) using specialized cameras

     • Track the centroid position of the gold nanoparticle

     • Convert linear displacements to angular movements

  Correlation with functional measurements:

  1. Simultaneous monitoring of rotation and proton translocation:

     • Combine fluorescence rotation tracking with pH-sensitive indicators

     • Correlate rotation events with proton movement

     • Determine the proton:step stoichiometry

  Technique	Temporal Resolution	Spatial Resolution	Environmental Compatibility	Key Information Obtained
  FRET	1-100 ms	1-10 nm	Compatible with membranes	Conformational changes, step size
  TIRF	5-30 ms	10-50 nm	Surface-tethered samples	Rotation rate, step size, dwell times
  HS-AFM	100-200 ms	1-2 nm	Native-like environments	Structural changes during rotation
  Gold nanoparticle	0.5-1 ms	5-10 nm	Various membranes	Precise angular movements, torque

  By applying these advanced biophysical approaches, researchers can gain detailed insights into the rotation dynamics of the c-ring, including step size, rotation rate, dwell times, and how these parameters respond to different conditions such as pH gradients, substrate concentrations, and inhibitors.

• How can site-directed mutagenesis of recombinant ATP synthase subunit c be used to investigate the mechanism of proton translocation?

  Site-directed mutagenesis of recombinant ATP synthase subunit c provides a powerful approach to dissect the molecular mechanism of proton translocation. Here is a comprehensive methodological framework for using this technique effectively:

  Key residues for mutagenesis:

  1. Proton-binding residue:

     • Identify the conserved acidic residue (glutamate or aspartate) that serves as the proton-binding site

     • Create mutations that alter proton affinity (E→D, E→Q, E→A)

     • Assess how these changes affect proton binding, pKa values, and translocation rates

  2. Surrounding polar/charged residues:

     • Target amino acids that form the proton translocation pathway

     • Investigate residues that may participate in hydrogen-bonding networks

     • Create charge-reversal mutations to understand electrostatic contributions

  Mutagenesis strategies:

  1. Alanine scanning:

     • Systematically replace individual residues with alanine

     • Assess the functional importance of each amino acid

     • Identify residues critical for proton binding, c-ring assembly, or rotation

  2. Conservative vs. non-conservative substitutions:

     • Compare effects of subtle changes (E→D) with more drastic ones (E→A, E→K)

     • Determine tolerance for different types of substitutions at key positions

  Functional assays for mutant characterization:

  1. Proton translocation measurements:

     • Reconstitute mutant proteins into liposomes

     • Use pH-sensitive fluorescent dyes (ACMA, pyranine) to monitor proton movement

     • Compare translocation rates and efficiency across mutants

  2. pH-dependent activity assays:

     • Determine how mutation affects the pH-dependence of activity

     • Construct pH profiles to identify pKa shifts of the proton-binding site

     • Correlate structural changes with altered pH sensitivity

  Mutation Type	Expected Effect	Experimental Readout	Interpretation
  E→D (conservative)	Altered pKa, maintained function	Shifted pH optimum	Proton affinity contribution
  E→Q (polar, non-protonable)	Loss of proton binding	Greatly reduced activity	Essential for proton binding
  E→A (non-polar, neutral)	Complete loss of function	No activity	Confirms essential role
  Neighboring polar→nonpolar	Disrupted H-bond network	Reduced efficiency	Supports proton wire model
  Interface residue mutations	Altered c-ring stability	Changed oligomeric state	Role in ring formation

  Structure-function correlation:

  1. Molecular dynamics simulations:

     • Model the effects of mutations on structure and dynamics

     • Simulate proton transfer events in wild-type and mutant systems

     • Identify compensatory mechanisms or altered proton pathways

  2. Correlation with chloroplast-specific features:

     • Investigate whether mutations in subunit c affect interaction with the redox-regulated γ subunit

     • Determine if proton translocation through mutant c-rings responds differently to light-dark transitions

     • Explore potential cross-talk between c-ring function and thioredoxin-mediated regulation

  By systematically applying these mutagenesis strategies and analytical approaches, researchers can develop a comprehensive understanding of the molecular mechanism underlying proton translocation through the ATP synthase c subunit and its coupling to rotational catalysis in the chloroplast environment.