Recombinant Lactuca sativa ATP synthase subunit c, chloroplastic (atpH)

What is the structure and function of ATP synthase subunit c (atpH) in Lactuca sativa chloroplasts?

ATP synthase subunit c (atpH) in Lactuca sativa is a small, highly hydrophobic membrane protein that forms part of the F₀ motor complex in chloroplast ATP synthase. This protein assembles into a ring structure in the thylakoid membrane that rotates during ATP synthesis. The c-subunit contains a critical glutamate residue that participates in proton translocation across the membrane, which drives the rotary mechanism of ATP synthesis. The protein's structure features two transmembrane α-helices connected by a short polar loop, with the conserved glutamate positioned to accept and release protons as the ring rotates .

The primary function of atpH is to convert the energy of the electrochemical proton gradient generated during photosynthesis into mechanical energy (rotation), which is then converted into chemical energy in the form of ATP by the F₁ catalytic head. This process is essential for energy production during photosynthesis in plants like Lactuca sativa .

What expression systems are most effective for producing recombinant Lactuca sativa atpH?

Based on successful approaches with homologous proteins, E. coli represents the most effective heterologous expression system for producing recombinant Lactuca sativa atpH. The bacterial expression system offers several advantages for this specific protein:

• High yield production of the relatively small (approximately 81 amino acid) protein

• Well-established protocols for membrane protein expression

• Compatibility with N-terminal His-tagging for simplified purification

• Cost-effectiveness and scalability for research purposes

When expressing Lactuca sativa atpH in E. coli, researchers should consider:

• Using specialized E. coli strains designed for membrane protein expression

• Optimizing codon usage for E. coli

• Employing inducible promoter systems (such as T7) with careful temperature control

• Including appropriate signal sequences if necessary for membrane insertion

For functional studies, alternative expression systems such as chloroplast-containing organisms (Chlamydomonas reinhardtii) might offer advantages for proper folding and assembly into functional complexes .

How do post-translational modifications affect the functionality of recombinant Lactuca sativa atpH in experimental settings?

Post-translational modifications (PTMs) of recombinant Lactuca sativa atpH can significantly impact its functionality in experimental settings. Unlike bacterial homologs, plant ATP synthase components often undergo specific PTMs that influence assembly, stability, and regulatory properties of the complex.

Key PTMs that may affect recombinant Lactuca sativa atpH include:

When using recombinant atpH for functional studies, researchers should compare the modification state of the recombinant protein with the native form using mass spectrometry. Differences in PTM patterns between recombinant and native proteins could explain functional discrepancies in experimental results. Additionally, reconstitution experiments should consider the lipid environment, as the c-subunit has specific lipid-binding properties that influence its assembly and function in the membrane environment .

What methodological approaches can resolve contradictory findings in proton translocation studies using recombinant Lactuca sativa atpH?

Contradictory findings in proton translocation studies involving recombinant Lactuca sativa atpH often stem from methodological variations. To resolve these contradictions, researchers should implement a multi-faceted approach:

• Standardized Reconstitution Protocol:

  • Use defined lipid compositions that mimic the thylakoid membrane

  • Control protein-to-lipid ratios precisely

  • Establish consistent buffer compositions with defined pH gradients

  • Verify complete reconstitution using freeze-fracture electron microscopy

• Multiple Complementary Measurement Techniques:

  • Combine fluorescence-based proton flux assays (ACMA fluorescence quenching)

  • Employ direct electrical measurements (solid-supported membrane electrophysiology)

  • Utilize isotope exchange experiments (H/D exchange mass spectrometry)

  • Perform structural studies (cryo-EM) in parallel with functional assays

• Refined Data Analysis:

  • Apply multiple mathematical models to raw data

  • Determine rate constants under varying conditions

  • Account for differences in reconstitution efficiency between preparations

A particularly effective approach is to systematically vary a single parameter (pH, membrane potential, lipid composition) while keeping all others constant, then construct a comprehensive model that predicts behavior across all conditions. This methodological rigor can identify whether contradictions arise from experimental artifacts or represent genuine alternative mechanisms of proton translocation through the c-ring structure .

How does the redox environment influence the assembly and function of recombinant Lactuca sativa atpH in experimental systems?

The redox environment critically influences both the assembly and function of recombinant Lactuca sativa atpH in experimental systems, particularly due to the redox-sensitive nature of chloroplast ATP synthase regulation.

In chloroplast ATP synthase, a conserved redox-controlled inhibitory element is present in the γ-subunit, which interacts with the c-ring during rotary catalysis. This redox switch, comprising a β-hairpin structure, blocks rotation in reducing conditions (dark) and permits rotation in oxidizing conditions (light). While this regulatory element is not located on atpH itself, the functional interaction between the c-ring and this element is critical for understanding the complete regulatory mechanism .

For experimental systems using recombinant Lactuca sativa atpH:

• Assembly Considerations:

  • Reconstitution experiments should control redox potential using defined ratios of oxidized/reduced glutathione

  • Assembly efficiency may differ significantly between reducing and oxidizing conditions

  • Co-reconstitution with other ATP synthase subunits should account for redox state

• Functional Assessment:

  • ATPase/ATP synthase activity assays should be performed under both reducing and oxidizing conditions

  • Light-dependent activation can be simulated by controlled redox transitions

  • Rotation assays may show distinct behaviors depending on redox environment

• Experimental Design Strategy:

  • Include thioredoxin systems when studying complete ATP synthase complexes

  • Compare behaviors in the presence of different redox mediators

  • Monitor real-time changes in activity during redox transitions

The interplay between redox regulation and proton-driven rotation makes chloroplast ATP synthase uniquely complex compared to bacterial or mitochondrial homologs, requiring careful consideration of redox conditions in all experimental designs involving recombinant Lactuca sativa atpH .

What purification protocol optimizations are necessary for high-yield isolation of functional recombinant Lactuca sativa atpH?

Purifying functional recombinant Lactuca sativa atpH requires specific optimizations to overcome the hydrophobic nature of this membrane protein while maintaining its structural integrity. Based on successful approaches with homologous proteins, the following protocol optimizations are recommended:

• Expression Optimization:

  • Induce expression at lower temperatures (16-20°C) to prevent inclusion body formation

  • Use specialized E. coli strains (C41/C43) designed for membrane protein expression

  • Employ auto-induction media for gradual protein expression

  • Harvest cells at optimal density (OD600 = 1.0-1.2) before membrane stress responses are triggered

• Membrane Extraction:

  • Use a two-step solubilization approach:
    a) Mild detergent (0.5% DDM) treatment to remove peripheral membrane proteins
    b) Stronger solubilization (1-2% DDM or 1% LDAO) for complete extraction

  • Include protease inhibitors and reducing agents throughout all steps

  • Perform extraction at 4°C with gentle agitation for 2-3 hours

• Affinity Purification:

  • For His-tagged constructs, use Co²⁺-based resins rather than Ni²⁺ for higher specificity

  • Apply low imidazole (10-20 mM) in wash buffers to minimize non-specific binding

  • Use gravity flow rather than pressure systems to prevent protein aggregation

  • Elute with step gradient of imidazole (50, 100, 200, 300 mM) to separate populations

• Detergent Exchange and Concentration:

  • Gradually exchange harsh solubilization detergents with milder ones (e.g., 0.03% DDM)

  • Use specialized concentration devices with low protein-binding membranes

  • Avoid concentrating beyond 1-2 mg/mL to prevent aggregation

  • Consider adding lipids (0.1-0.2 mg/mL) during concentration to stabilize protein

• Quality Assessment:

  • Perform size-exclusion chromatography to verify monodispersity

  • Use circular dichroism to confirm α-helical secondary structure

  • Verify purity by SDS-PAGE with specialized membrane protein staining techniques

Typical yield from optimized protocols can reach 1-3 mg of purified protein per liter of E. coli culture, with >90% purity as assessed by SDS-PAGE .

What reconstitution methods best preserve the functional characteristics of Lactuca sativa atpH for biophysical studies?

For biophysical studies of Lactuca sativa atpH, reconstitution methods must balance protein stability with the creation of a native-like membrane environment. The following methodologies have been demonstrated to effectively preserve functional characteristics:

• Detergent-Mediated Reconstitution:

  • Start with purified protein in 0.03% DDM or 0.1% LDAO

  • Use a lipid mixture mimicking thylakoid composition (MGDG/DGDG/SQDG/PG at 40:30:15:15 ratio)

  • Prepare unilamellar liposomes (100-200 nm) by extrusion

  • Add detergent-solubilized protein to destabilized liposomes (protein:lipid ratio 1:100-1:200)

  • Remove detergent by adsorption to Bio-Beads SM-2 or dialysis against detergent-free buffer

  • Verify reconstitution by freeze-fracture electron microscopy or dynamic light scattering

• Direct Incorporation into Nanodiscs:

  • Select MSP1D1 or MSP1E3D1 scaffold proteins based on the desired nanodisc size

  • Mix purified atpH, MSP protein, and lipids at optimized ratios (typically 1:2:60-120)

  • Remove detergent gradually using Bio-Beads at 4°C overnight

  • Purify assembled nanodiscs by size-exclusion chromatography

  • This approach provides a more defined membrane environment and is particularly suitable for single-molecule studies

• Functional Verification Methods:

  • Proton translocation assay using pH-sensitive fluorescent dyes (ACMA)

  • Circular dichroism spectroscopy to confirm secondary structure integrity

  • Differential scanning calorimetry to assess thermal stability

  • Native mass spectrometry to verify complex formation

• Critical Parameters for Functional Preservation:

These reconstitution approaches provide complementary advantages, with liposome reconstitution better suited for bulk functional assays and nanodiscs preferred for high-resolution structural studies or single-molecule biophysics .

How can researchers accurately assess the orientation of reconstituted Lactuca sativa atpH in membrane systems?

Determining the orientation of reconstituted Lactuca sativa atpH in membrane systems is crucial for interpreting functional data correctly, as the protein must maintain its native topology for proper proton translocation. The following methodological approaches provide accurate assessment of protein orientation:

• Protease Accessibility Assays:

  • Introduce specific protease cleavage sites at solvent-exposed loops

  • Treat intact proteoliposomes with membrane-impermeable proteases

  • Analyze fragmentation patterns by SDS-PAGE or mass spectrometry

  • Compare results with detergent-permeabilized samples to differentiate inside-out vs. right-side-out orientations

• Antibody-Based Detection:

  • Generate antibodies against epitopes on specific domains of the protein

  • Perform immunolabeling without membrane permeabilization

  • Quantify binding using flow cytometry or ELISA techniques

  • Calculate the percentage of correctly oriented protein

• Fluorescence Quenching Techniques:

  • Introduce single cysteine residues at strategic positions

  • Label with environment-sensitive fluorophores (e.g., IAEDANS)

  • Measure fluorescence quenching by membrane-impermeable quenchers

  • The quenching efficiency correlates with the exposure of labeled sites

• Electron Microscopy with Gold Labeling:

  • Attach gold nanoparticles to specific domains using antibodies or nickel-NTA chemistry

  • Visualize labeled proteoliposomes using transmission electron microscopy

  • Determine orientation based on the position of gold particles relative to the membrane

• Functional Orientation Assessment:

  • Incorporate pH-sensitive fluorescent probes inside liposomes

  • Create a pH gradient across the membrane

  • Measure proton translocation activity in response to ATP or an imposed membrane potential

  • Directional proton movement indicates the functional orientation of the c-ring

Quantitative analysis of these results typically shows a mixed population, with 60-70% of proteins incorporating in the correct orientation under optimal reconstitution conditions. This asymmetry should be accounted for when calculating kinetic parameters from bulk measurements .

What statistical approaches are most appropriate for analyzing rotational catalysis data from single-molecule studies of Lactuca sativa atpH?

Single-molecule studies of rotational catalysis in Lactuca sativa atpH require specialized statistical approaches to extract meaningful information from inherently noisy data. The following statistical methods are most appropriate for this research:

• Hidden Markov Model (HMM) Analysis:

  • Ideal for identifying discrete states in stepping rotation data

  • Estimates transition probabilities between states

  • Models the underlying step size distribution

  • Implementation example: vbFRET or HaMMy software packages

  • Particularly useful for detecting substeps in c-ring rotation

• Dwell Time Distribution Analysis:

  • Fits dwell time histograms to appropriate probability distributions

  • Usually employs gamma distribution for multi-exponential processes

  • Reveals the number of rate-limiting steps in the catalytic cycle

  • Can be used to extract forward and backward rate constants

  • Critical for determining the coupling ratio between proton translocation and ATP synthesis

• Power Spectrum Analysis:

  • Transforms time-domain data to frequency domain

  • Identifies characteristic frequencies in rotational motion

  • Separates signal from noise based on frequency components

  • Useful for detecting periodic patterns in noisy datasets

• Bayesian Change Point Detection:

  • Identifies points where statistical properties change significantly

  • More robust than threshold-based detection for noisy biological data

  • Provides confidence intervals for detected steps

  • Particularly valuable for analyzing data at limiting ATP concentrations

• Bootstrap Resampling for Error Estimation:

  • Generates synthetic datasets by resampling with replacement

  • Provides robust error estimates for fitted parameters

  • Essential for comparing results between different experimental conditions

  • Recommended minimum: 1000 bootstrap iterations for parameter estimation

A comprehensive analytical approach would combine these methods in a sequential workflow:

• Preprocess raw data using moving average or Chung-Kennedy filters

• Apply HMM to identify discrete states

• Perform dwell time analysis on segmented data

• Use bootstrap resampling to estimate parameter confidence intervals

• Validate results by comparing to simulated data with known parameters

This multilayered statistical approach minimizes the risk of misinterpreting artifacts as biological signals and provides robust quantification of rotational dynamics .

How can researchers accurately determine the stoichiometry of proton translocation to ATP synthesis in systems containing recombinant Lactuca sativa atpH?

Determining the precise stoichiometry of proton translocation to ATP synthesis (H⁺/ATP ratio) in systems containing recombinant Lactuca sativa atpH requires multi-methodological approaches to overcome experimental limitations. The following integrated strategy provides the most accurate determination:

• Structural Analysis of c-ring Composition:

  • High-resolution cryo-EM or X-ray crystallography to determine the number of c-subunits per ring

  • Mass spectrometry of intact complexes to confirm subunit stoichiometry

  • Each c-subunit contains one proton-binding site, so the number of c-subunits equals the H⁺ translocated per 360° rotation

• Thermodynamic Equilibrium Measurements:

  • Create defined proton gradients (ΔpH) and membrane potentials (Δψ)

  • Measure ATP/ADP ratios at equilibrium (no net synthesis or hydrolysis)

  • Calculate H⁺/ATP from the relationship: n = ΔG(ATP synthesis)/ΔμH⁺

  • This approach is independent of kinetic complications

• Real-time Measurements of Coupled Processes:

  • Simultaneous monitoring of:

    • Proton uptake/release (pH-sensitive dyes)

    • ATP synthesis/hydrolysis (luciferase assay or coupled enzyme assays)

    • Membrane potential (voltage-sensitive dyes)

  • Plot the relationship between rates at various driving forces

• Single-Molecule Rotation Analysis:

  • Attach fluorescent probes to the γ subunit to visualize rotation

  • Correlate stepwise rotation with ATP binding/hydrolysis events

  • The number of steps per 360° rotation corresponds to the ATP synthesized per full rotation

• Data Integration Approach:

Based on these complementary approaches, the consensus H⁺/ATP ratio for chloroplast ATP synthase containing Lactuca sativa atpH is approximately 4.67, reflecting a c-ring with 14 subunits and synthesis of 3 ATP molecules per complete rotation. This ratio is higher than in mitochondrial ATP synthase (2.7-3.3), reflecting adaptation to the different energetic constraints of photosynthesis versus respiration .

What approaches can distinguish between specific inhibition of Lactuca sativa atpH function and general disruption of membrane integrity in experimental assays?

Distinguishing between specific inhibition of Lactuca sativa atpH function and non-specific membrane disruption is crucial for accurate interpretation of inhibition studies. The following methodological approaches provide the necessary differentiation:

• Parallel Membrane Integrity Assays:

  • Perform calcein leakage assays in parallel with functional studies

  • Monitor changes in membrane potential using DiSC3(5) fluorescence

  • Measure light scattering to detect gross morphological changes

  • These controls should be negative while atpH function is inhibited

• Specific Activity Measurements:

  • Compare ATP synthesis inhibition with proton translocation inhibition

  • Use reconstituted systems with defined composition

  • Include respiratory chain components as internal controls

  • Specific inhibition should affect ATP synthase activity without affecting control proteins

• Site-Directed Mutagenesis Approach:

  • Generate point mutations at proposed inhibitor binding sites

  • Compare inhibition profiles between wild-type and mutant proteins

  • Reduced sensitivity in mutants confirms target specificity

  • Maintain catalog of resistance mutations for different inhibitors

• Binding Assays:

  • Measure direct binding of inhibitors to purified protein

  • Use techniques such as isothermal titration calorimetry or microscale thermophoresis

  • Correlate binding constants with functional inhibition

  • Competition assays with known inhibitors provide additional verification

• Decision Matrix for Inhibition Mechanism:

• Computational Validation:

  • Molecular docking simulations to predict binding sites

  • Molecular dynamics to assess effects on protein structure

  • Comparison with experimentally derived structure-activity relationships

  • This approach is particularly valuable for designing control experiments

By implementing this multi-faceted approach, researchers can confidently distinguish between genuine inhibitors of Lactuca sativa atpH function and compounds that merely disrupt membrane integrity, leading to more reliable identification of specific inhibitors and their mechanisms of action .

What strategies can resolve expression challenges when producing recombinant Lactuca sativa atpH in bacterial systems?

Expression of recombinant Lactuca sativa atpH in bacterial systems frequently encounters challenges due to its hydrophobic nature and potential toxicity. The following strategic interventions can effectively resolve these issues:

• Addressing Toxicity Issues:

  • Use tightly controlled inducible promoters (e.g., pBAD or tet-inducible systems)

  • Maintain glucose repression until induction point

  • Employ specialized E. coli strains (C41/C43) designed for toxic membrane proteins

  • Consider cell-free expression systems for highly toxic constructs

• Improving Protein Folding:

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Include low concentrations of non-denaturing detergents in growth media

  • Optimize growth temperature (typically 16-20°C after induction)

  • Add chemical chaperones such as glycerol (5-10%) to culture medium

• Enhancing Membrane Integration:

  • Co-express with membrane integrase proteins

  • Optimize the signal sequence for bacterial expression

  • Consider fusion partners that assist membrane targeting

  • Culture cells in media supplemented with specific phospholipids

• Genetic Optimization Strategies:

  • Codon optimization for E. coli expression

  • Remove rare codons or potential internal ribosome binding sites

  • Introduce solubility-enhancing mutations without affecting function

  • Use dual vector systems for coordinated expression of multiple components

• Systematic Troubleshooting Guide:

• Expression Construct Design Guidelines:

  • Include C-terminal rather than N-terminal tags when possible

  • Use small affinity tags (His6 rather than larger tags)

  • Consider dual tagging for verification of full-length expression

  • Include TEV cleavage sites for tag removal after purification

Implementing these strategies systematically can increase expression yield from undetectable levels to 1-3 mg/L culture, making subsequent purification and functional studies feasible .

What approaches can minimize protein aggregation during purification and storage of recombinant Lactuca sativa atpH?

Protein aggregation represents a major challenge in working with recombinant Lactuca sativa atpH due to its hydrophobic nature. The following comprehensive strategies can effectively minimize aggregation during purification and storage:

• Optimized Detergent Selection:

  • Conduct detergent screening using thermal stability assays

  • Consider mild detergents for final buffer (DDM, LMNG, GDN)

  • Use detergent concentrations just above critical micelle concentration

  • Typical optimal detergents for atpH stability:

• Buffer Optimization:

  • Maintain pH between 7.0-8.0 to prevent isoelectric precipitation

  • Include glycerol (10-20%) as molecular crowding agent

  • Add specific lipids (0.1-0.2 mg/mL DGDG or SQDG) to stabilize structure

  • Incorporate mild reducing agents (1-2 mM DTT or 5 mM β-mercaptoethanol)

  • Use higher salt concentration (200-300 mM NaCl) to screen electrostatic interactions

• Temperature Management:

  • Maintain all purification steps at 4°C

  • Avoid freeze-thaw cycles by aliquoting before freezing

  • For storage, flash-freeze in liquid nitrogen rather than slow freezing

  • Consider storage at -80°C rather than -20°C for long-term stability

• Purification Workflow Modifications:

  • Use gravity flow rather than high pressure in chromatography

  • Elute proteins with shallow gradients to prevent concentration spikes

  • Include short centrifugation steps (100,000×g, 10 min) before each chromatography step

  • Limit protein concentration to <5 mg/mL during purification

• Advanced Stabilization Techniques:

  • Amphipol exchange for detergent removal

  • Nanodisc incorporation for membrane-like environment

  • Complex formation with stabilizing partner proteins

  • Addition of specific ligands or inhibitors that stabilize conformation

• Long-term Storage Recommendations:

  • Store at protein concentration of 1-2 mg/mL

  • Add trehalose (6%) as cryoprotectant

  • Store as multiple small aliquots to avoid repeated thawing

  • For maximum stability, consider lyophilization in the presence of disaccharides

  • Monitor protein quality by analytical SEC after storage periods

• Real-time Aggregation Monitoring:

  • Implement dynamic light scattering before and after storage

  • Use fluorescence-based thermal shift assays to assess stability

  • Monitor tryptophan fluorescence to detect conformational changes

  • These techniques provide early warning of aggregation tendency

By implementing these complementary approaches, researchers can typically extend the functional lifetime of purified recombinant Lactuca sativa atpH from days to several months, greatly facilitating downstream structural and functional studies .