Recombinant Aethionema cordifolium ATP synthase subunit c, chloroplastic (atpH)

What is ATP synthase subunit c in chloroplasts and what is its significance in photosynthetic research?

ATP synthase subunit c (atpH) is a critical component of the chloroplastic ATP synthase complex responsible for ATP production during photosynthesis. This protein forms the c-ring structure embedded in the thylakoid membrane, which plays a central role in the mechanical coupling between proton translocation and ATP synthesis. In chloroplasts, the multimeric ATP synthase produces the adenosine triphosphate (ATP) required for photosynthetic metabolism, with the c-ring rotation driven by proton translocation across the thylakoid membrane along an electrochemical gradient .

The significance of studying this protein lies in understanding the fundamental mechanisms of photosynthetic energy conversion. The rotation of the c-ring is coupled to the rotation of the γ-stalk in the F₁ region, driving the catalysis of ADP + Pi → ATP at the three α-β subunit interfaces. This cyclical sequence of rotation, translocation, and catalysis produces 3 ATP molecules for every n protons that pass from the lumen to the stroma, where n represents the number of c-subunits in the ring .

What expression systems are most effective for recombinant production of chloroplastic ATP synthase subunit c?

The most effective expression systems for recombinant ATP synthase subunit c production utilize Escherichia coli with specialized vectors designed to enhance expression and solubility of this membrane protein. Based on comparative experimental data, the pMAL-c2x vector system with the maltose-binding protein (MBP) fusion tag has demonstrated superior results for chloroplastic ATP synthase subunit c expression .

Several expression systems can be evaluated for recombinant ATP synthase subunit c production:

The pMAL system with MBP fusion has proven particularly effective because MBP enhances solubility of the hydrophobic c-subunit. Co-expression with chaperone proteins (DnaK, DnaJ, and GrpE) can substantially increase quantities of recombinant proteins that are toxic or otherwise difficult to produce .

How should the atpH gene be cloned for optimal recombinant expression?

For optimal recombinant expression of the atpH gene, the following methodological approach is recommended:

• Gene synthesis and optimization: The atpH gene should be synthesized with codon optimization for the host expression system (typically E. coli). Codon optimization can significantly enhance expression levels by matching codon usage to the host's preferred codons .

• Strategic restriction site design: Incorporate appropriate restriction sites at the 5' and 3' ends of the gene for directional cloning. For example, NdeI at the 5' end and XhoI at the 3' end work well with many expression vectors .

• Vector selection and preparation: Select a vector that allows fusion with a solubility-enhancing tag. The pMAL-c2x vector system has proven effective, allowing insertion at XmnI and XhoI restriction sites to produce the plasmid pMAL-c2x-malE/atpH .

• Cloning procedure:

  • Amplify the synthetic atpH gene using high-fidelity PCR with a proofreading polymerase such as Phusion Polymerase

  • Digest both the PCR product and vector with appropriate restriction enzymes

  • Ligate the digested gene into the prepared vector

  • Transform into a cloning strain of E. coli

  • Verify the construct by sequencing before transforming into an expression strain

• Expression strain selection: Transform the verified construct into an expression-optimized E. coli strain such as T7 Express lysY/Iᵍ for controlled, high-level expression .

What purification strategies yield highly pure ATP synthase subunit c protein?

Purification of recombinant ATP synthase subunit c requires a multi-step approach to obtain highly pure protein suitable for structural and functional studies:

• Initial clarification: After cell lysis, centrifugation at 20,000 × g separates soluble proteins from cell debris. For membrane-associated proteins like ATP synthase subunit c, detergent solubilization may be necessary using mild detergents such as n-dodecyl-β-D-maltoside (DDM) or n-octyl-β-D-glucopyranoside (OG) .

• Affinity chromatography: For MBP fusion constructs, amylose resin affinity chromatography provides the first purification step. The fusion protein binds to the amylose resin and can be eluted with maltose-containing buffer (typically 10 mM maltose) .

• Tag removal: Cleave the fusion tag using a specific protease (e.g., Factor Xa for MBP fusion) under optimized conditions. The cleavage reaction typically requires:

  • Buffer optimization (pH 7.5-8.0)

  • Calcium ions (1-2 mM)

  • Controlled temperature (20-25°C)

  • Optimized enzyme:substrate ratio

• Secondary purification: Following tag removal, apply reverse affinity chromatography to separate the cleaved target protein from the fusion tag. Size exclusion chromatography (SEC) provides further purification and simultaneous buffer exchange .

• Final polishing: Ion exchange chromatography can be employed as a final polishing step to remove any remaining contaminants, taking advantage of the protein's isoelectric point (pI) .

Quality control at each purification step is essential, using SDS-PAGE analysis to assess purity and Western blotting to confirm identity. The purified protein should be analyzed for correct alpha-helical secondary structure using circular dichroism spectroscopy .

How can I verify the structural integrity of purified recombinant ATP synthase subunit c?

Verifying the structural integrity of purified recombinant ATP synthase subunit c requires multiple complementary analytical techniques:

• Circular Dichroism (CD) Spectroscopy: This technique provides essential information about the secondary structure of the protein. ATP synthase subunit c should exhibit the correct alpha-helical secondary structure, characterized by negative bands at 208 nm and 222 nm in the CD spectrum. Comparing the CD spectrum of your recombinant protein with published data for native ATP synthase subunit c can confirm proper folding .

• Size Exclusion Chromatography (SEC): SEC can determine the oligomeric state and homogeneity of the purified protein. The elution volume should correspond to the expected molecular weight of the monomeric or oligomeric state, depending on solubilization conditions .

• Dynamic Light Scattering (DLS): DLS provides information about the size distribution and potential aggregation of the purified protein. A monodisperse population indicates properly folded protein, while polydispersity may suggest partial unfolding or aggregation .

• Limited Proteolysis: Controlled digestion with proteases can reveal the compactness and domain organization of the protein. Well-folded proteins show resistance to proteolysis compared to unfolded ones. The pH-dependence of protease susceptibility should also be investigated, as crystal growth often occurs at different pH values than functional studies .

• Functional Assays: Activity assays that measure proton translocation capability or reconstitution into liposomes can confirm functional integrity. Comparison of activity with the native protein provides a benchmark for biological relevance .

How does the stoichiometry of c-subunits in the c-ring affect ATP synthesis efficiency in chloroplasts?

The stoichiometry of c-subunits in the ATP synthase c-ring directly impacts the bioenergetic efficiency of ATP synthesis in chloroplasts. This relationship stems from the fundamental mechanism of ATP synthesis:

The ratio of protons translocated to ATP synthesized varies according to the number of c-subunits (n) in the ring. For each complete rotation of the c-ring, which requires n protons to translocate across the membrane, 3 ATP molecules are synthesized in the F₁ catalytic domain. Therefore, the H⁺/ATP ratio equals n/3 .

Methodological approaches to investigate this relationship include:

• Comparative structural analysis: Determine c-ring stoichiometry across different species using cryo-electron microscopy or X-ray crystallography. This requires:

  • High-purity recombinant protein preparation

  • Optimization of detergent conditions

  • Screening of crystallization conditions with varying pH, temperature, and precipitants

• Site-directed mutagenesis: Introduce mutations at the c-subunit interfaces to alter packing preferences and potentially modify ring stoichiometry. Key residues at the interface between adjacent c-subunits can be identified through sequence conservation analysis across species .

• Biophysical characterization: Use analytical ultracentrifugation, size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), and native mass spectrometry to determine the precise molecular weight of the assembled c-ring, which directly correlates with stoichiometry .

• Functional reconstitution studies: Reconstitute c-rings with varying stoichiometries into liposomes and measure ATP synthesis rates and proton translocation efficiency under different proton motive force conditions. This requires:

  • Purification of additional ATP synthase components

  • Controlled liposome preparation with defined lipid composition

  • Precise measurement of ATP synthesis using luciferase-based assays

The evolutionary significance of variable c-ring stoichiometry likely reflects adaptation to different energetic constraints across species and cellular compartments. In chloroplasts, the typically higher number of c-subunits (14 in spinach) compared to mitochondria (8-10) may represent adaptation to the different magnitude of proton motive force available in these organelles .

What are the critical challenges in crystallizing recombinant ATP synthase subunit c for structural studies?

Crystallizing recombinant ATP synthase subunit c presents several significant challenges that must be addressed through methodical approaches:

• Membrane protein solubilization: ATP synthase subunit c is highly hydrophobic with two transmembrane helices. Selecting appropriate detergents is critical:

  • Screen multiple detergents (DDM, OG, LDAO, C₁₂E₈)

  • Optimize detergent concentration to maintain protein stability while avoiding micelle formation that interferes with crystal contacts

  • Consider novel solubilization agents such as amphipols or nanodiscs for improved stability

• pH-dependent conformational changes: ATP synthase components can undergo significant conformational changes with pH variations. Experimental design should include:

  • Screening crystallization conditions across a wide pH range (pH 5-9)

  • Investigating pH-dependent structural changes using limited proteolysis at various pH values

  • Monitoring secondary structure stability across pH range using circular dichroism

• Protein homogeneity challenges: Achieving homogeneous protein preparations is essential for crystallization:

  • Employ multiple purification steps including reverse affinity chromatography after tag cleavage

  • Use size exclusion chromatography as final polishing step

  • Verify monodispersity using dynamic light scattering before crystallization trials

• Crystal packing constraints: The cylindrical shape of assembled c-rings creates packing challenges:

  • Screen additives that promote specific crystal contacts

  • Try both vapor diffusion and lipidic cubic phase crystallization methods

  • Consider crystallizing the monomeric form with fusion partners that promote crystallization

• Data collection and processing: Once crystals are obtained, data collection presents additional challenges:

  • Radiation damage is significant for membrane protein crystals

  • Multiple crystals may be needed to obtain complete datasets

  • Data processing requires careful handling of anisotropic diffraction

Successful crystallization typically requires hundreds of conditions and iterative optimization. The reported unit cell parameters (a = 144.0, c = 351.2 Å) and space group (H3, hexagonal setting of R3) from related ATP synthase component structures provide starting parameters for experimental design and data processing .

What molecular biology techniques can be applied to investigate structure-function relationships in ATP synthase subunit c?

Advanced molecular biology techniques can reveal critical structure-function relationships in ATP synthase subunit c:

• Site-directed mutagenesis: Target specific residues to investigate their roles in c-ring assembly, proton translocation, and interaction with other ATP synthase components:

  • Conserved glutamate/aspartate residues in the middle of the second transmembrane helix are essential for proton binding/release

  • Interface residues between adjacent c-subunits affect c-ring stability and stoichiometry

  • Use a systematic alanine-scanning approach to identify critical residues

• Chimeric protein construction: Create fusion proteins between c-subunits from different species to investigate domain-specific functions:

  • Swap transmembrane domains between species with different c-ring stoichiometries

  • Use specialized cloning techniques such as Gibson Assembly or FastCloning for seamless domain exchanges

  • Express in the pMAL-c2x system with MBP fusion for enhanced solubility

• Cysteine-scanning mutagenesis combined with site-specific labeling:

  • Introduce single cysteine residues at various positions

  • Label with fluorescent or spin-label probes

  • Monitor conformational changes during proton translocation using FRET or EPR spectroscopy

• Cross-linking studies to investigate protein-protein interactions:

  • Introduce paired cysteine residues at predicted interaction sites

  • Induce disulfide bond formation under oxidizing conditions

  • Analyze crosslinked products by SDS-PAGE and mass spectrometry

• Reconstitution studies with modified components:

  • Express and purify individual ATP synthase components

  • Reconstitute with wild-type and mutant c-subunits

  • Measure ATP synthesis rates to correlate structural changes with functional outcomes

The methodological workflow typically involves:
a) Design and creation of mutant constructs using PCR-based mutagenesis
b) Cloning into expression vectors such as pMAL-c2x-malE/atpH
c) Expression in E. coli T7 Express lysY/Iᵍ or similar strains
d) Purification using protocols optimized for ATP synthase subunit c
e) Structural characterization using CD spectroscopy to verify folding
f) Functional analysis in reconstituted systems

Conservation analysis across species can guide selection of target residues, with highly conserved residues between APP and APLP2 proteins suggesting functional importance at protein interfaces .

How can recombinant ATP synthase subunit c be incorporated into artificial membrane systems for functional studies?

Incorporating recombinant ATP synthase subunit c into artificial membrane systems enables detailed functional studies and requires several sophisticated methodological approaches:

• Liposome reconstitution:

  • Prepare liposomes using a defined lipid composition that mimics the thylakoid membrane (typically DOPC, DOPE, and DOPG at specified ratios)

  • Solubilize purified recombinant ATP synthase subunit c in appropriate detergent (C₁₂E₈ or DDM at 0.1-0.5%)

  • Mix protein and liposomes at protein:lipid ratios between 1:50 and 1:200 (w/w)

  • Remove detergent gradually using Bio-Beads SM-2 or controlled dialysis

  • Separate protein-containing proteoliposomes from empty liposomes using sucrose gradient centrifugation

• Planar lipid bilayer studies:

  • Form planar lipid bilayers across apertures in Teflon chambers

  • Incorporate purified c-rings using fusion of proteoliposomes or direct addition of detergent-solubilized protein

  • Measure ion conductance using patch-clamp techniques

  • Determine ion selectivity by varying ion compositions and measuring current-voltage relationships

• Nanodiscs assembly:

  • Mix purified recombinant ATP synthase subunit c with membrane scaffold proteins (MSPs) and appropriate lipids

  • Remove detergent using Bio-Beads to initiate self-assembly

  • Purify nanodiscs containing c-rings using size exclusion chromatography

  • Verify incorporation and orientation using electron microscopy and biochemical assays

• Functional assays in reconstituted systems:

  • Proton translocation: Monitor pH changes using pH-sensitive fluorescent dyes (ACMA or pyranine)

  • ATP synthesis: Measure ATP production in reconstituted systems using luciferin/luciferase assays

  • Rotation assays: Attach fluorescent probes to allow single-molecule visualization of c-ring rotation

• Co-reconstitution with other ATP synthase components:

  • Purify additional components of ATP synthase (F₁ complex, a/b subunits)

  • Reconstitute complete or partial ATP synthase complexes

  • Compare functionality between systems containing wild-type and modified c-subunits

The critical parameters for successful reconstitution include:

• Protein:lipid ratio optimization

• Careful detergent selection and removal rates

• pH and ionic strength control during reconstitution

• Verification of protein orientation within the membrane

These artificial membrane systems allow investigation of fundamental questions about c-ring function, including proton translocation mechanisms, coupling between c-ring rotation and ATP synthesis, and effects of mutations on functional properties.

What are the optimal conditions for analyzing c-ring assembly from recombinant monomeric ATP synthase subunit c?

Analyzing c-ring assembly from recombinant monomeric ATP synthase subunit c requires carefully optimized conditions and sophisticated analytical techniques:

• Buffer composition optimization:

  • pH range testing (pH 5-8): Monitor assembly at different pH values, as pH can significantly influence c-subunit oligomerization. Note that at acidic pH (5.7), addition of at least 500 mM NaCl is necessary for proper size-dependent retention in gel filtration chromatography

  • Ionic strength variation: Test NaCl concentrations from 50-500 mM to identify optimal conditions for stable c-ring formation

  • Divalent cations: Supplement with Mg²⁺ (1-10 mM) which can stabilize protein-protein interactions in the c-ring

• Detergent selection and concentration:

  • Screen multiple detergents (DDM, OG, LDAO, C₁₂E₈) at concentrations slightly above their critical micelle concentration (CMC)

  • Evaluate detergent effects on assembly kinetics and stability using analytical ultracentrifugation

  • Consider novel solubilization agents such as amphipols or SMA copolymers which may better preserve native interactions

• Analytical methods for monitoring assembly:

  • Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine absolute molecular weight with precision

  • GPC with calculated MWs based on retention volume for initial screening

  • Native PAGE with appropriate detergent systems to visualize oligomeric states

  • Basic light scattering to monitor molecular radius (MWrh) and SEC retention volume to calculate absolute molecular weight (MWabs)

• Assembly kinetics monitoring:

  • Time-course experiments sampling at regular intervals (0, 1, 3, 6, 12, 24, 48 hours)

  • Temperature variation (4°C, 25°C, 37°C) to determine optimal assembly conditions

  • Stopped-flow light scattering for real-time assembly monitoring

• Stabilizing factors for c-ring assembly:

  • Addition of specific lipids (cardiolipin, PG) that may facilitate c-ring formation

  • Evaluation of the effect of heparin and other polyanionic molecules that can induce dimerization or higher-order assemblies

  • pH-dependent stability analysis to identify conditions for maximal stability

The detailed analysis should include:

• Precise molecular weight determination using both GPC and SLS techniques (as shown in search result )

• Comparison between theoretical and measured masses to determine stoichiometry

• Electron microscopy validation of ring formation and structural integrity

Experimental data indicates that conditions strongly affect oligomeric state, with factors like pH, ionic strength, and the presence of polyanionic molecules like heparin playing crucial roles in assembly equilibria .

How do pH conditions affect structure and function of recombinant ATP synthase subunit c?

pH conditions significantly impact both the structure and function of recombinant ATP synthase subunit c through multiple mechanisms that must be carefully considered in experimental design:

• pH-dependent protonation states: The conserved carboxylate residue (Asp or Glu) in transmembrane helix 2 of subunit c undergoes protonation/deprotonation during the catalytic cycle. This residue has:

  • pKa ~7.0 in the lipid-exposed state

  • pKa ~5.0 when facing the a-subunit

  • Different experimental pH values can lock this residue in specific protonation states

• Structural stability across pH range: Recombinant ATP synthase subunit c exhibits varying stability at different pH values:

  • At acidic pH (5.0-6.0): Enhanced stability of the c-ring structure observed

  • At neutral pH (6.5-7.5): Optimal for functional studies but may affect assembly

  • At alkaline pH (>8.0): Increased susceptibility to limited proteolysis

• Experimental considerations for different pH conditions:

  • Crystallization: Crystal formation is often more successful at slightly acidic pH (5.5-6.5)

  • Functional studies: ATP synthesis assays typically performed at physiological pH (7.5-8.0)

  • Limited proteolysis data obtained at pH 8.0 may not reflect structural properties at crystallization pH, necessitating parallel experiments

• Buffer system selection:

  • For acidic range (pH 4.5-6.5): Citrate, MES, or acetate buffers

  • For neutral range (pH 6.5-7.5): MOPS or HEPES buffers

  • For alkaline range (pH 7.5-9.0): Tris or BICINE buffers

  • All at 20-50 mM concentration with appropriate ionic strength adjustment

• pH-dependent protein-protein interactions:

  • At pH 5.7, a minimum of 500 mM NaCl must be added to obtain proper size-dependent retention volume on GPC column materials, which unfortunately interferes with certain binding studies

  • Heparin binding to protein complexes can be pH-dependent, affecting oligomerization behavior

Methodological approach for pH studies:

• Prepare identical protein samples in buffers spanning pH 5.0-9.0

• Analyze secondary structure stability using circular dichroism

• Perform limited proteolysis at each pH point

• Monitor oligomeric state using SEC-MALS

• Compare functional parameters (if applicable) across pH range

These studies are crucial for establishing optimal conditions for structural and functional experiments with recombinant ATP synthase subunit c.

What strategies can address expression challenges for recombinant chloroplastic proteins like ATP synthase subunit c?

Expressing recombinant chloroplastic proteins like ATP synthase subunit c presents several challenges that require strategic approaches:

• Codon optimization for host expression:

  • Analyze the codon usage bias of the target atpH gene from Aethionema cordifolium

  • Optimize codons to match preferred E. coli codons while maintaining GC content

  • Remove rare codons that might cause translational pausing or early termination

  • Eliminate problematic secondary structures in the mRNA that could impede translation

• Fusion tag selection for enhanced expression and solubility:

  • MBP fusion (via pMAL-c2x vector) significantly enhances solubility of membrane proteins like ATP synthase subunit c

  • Alternative tags like thioredoxin (Trx) from pET-32a(+) can assist proper folding

  • FLAG tag (pFLAG-MAC) provides easy detection but may not enhance solubility

  • Comparative testing of multiple fusion constructs is recommended for optimization

• Co-expression with molecular chaperones:

  • Co-transform expression cells with the pOFXT7KJE3 vector expressing DnaK, DnaJ, and GrpE chaperones

  • These chaperones substantially increase quantities of recombinant proteins that are toxic or otherwise difficult to produce

  • Optimize induction conditions for both the target protein and chaperones

• Expression conditions optimization:

  • Temperature: Lower temperatures (16-25°C) often improve folding of difficult proteins

  • Induction time: Extended expression periods at lower temperatures (16-24h)

  • Inducer concentration: Reduced IPTG concentrations (0.1-0.5 mM) for slower, more controlled expression

  • Media composition: Rich media (TB, 2xYT) or defined media supplements

• Specialized expression strains:

  • T7 Express lysY/Iᵍ strain controls basal expression through regulated T7 RNA polymerase

  • C41(DE3) and C43(DE3) strains are specifically adapted for toxic and membrane protein expression

  • Rosetta strains supply rare tRNAs that might be limiting for plant protein expression

Experimental approach:

• Design multiple constructs in parallel with different tags and promoter strengths

• Test small-scale expression (100 mL cultures) before scaling up

• Monitor expression using SDS-PAGE and Western blotting

• Quantify soluble vs. insoluble fraction distribution

• Optimize based on yield and solubility results

The pMAL-c2x-malE/atpH construct has demonstrated successful expression in E. coli, with significant enhancement when co-expressed with chaperone proteins from the pOFXT7KJE3 vector, making this an excellent starting point for recombinant ATP synthase subunit c expression .

What phylogenetic markers can be used alongside atpH gene to confirm evolutionary relationships in plant species?

When studying evolutionary relationships in plant species using the atpH gene, researchers should employ multiple chloroplast markers for robust phylogenetic analysis:

• Established barcoding loci that complement atpH:

  • rbcL gene: Coding for the large subunit of RuBisCO, this gene evolves relatively slowly and is useful for resolving relationships at higher taxonomic levels

  • matK gene: This gene evolves more rapidly than rbcL and provides resolution at lower taxonomic levels

  • trnH-psbA intergenic spacer: Highly variable region useful for species-level identification

  • rpoB and rpoC1: RNA polymerase genes that serve as effective control markers alongside atpH

• Additional high-variability chloroplast markers:

  • rps16-trnQ: Demonstrated high variability across diverse plant lineages

  • trnK: Contains the matK gene and provides additional sequence information

  • trnS-UGA-trnG-UCC: Intergenic spacer with high discriminatory power

  • ycf1: Although challenging for primer design due to length and variability, provides excellent phylogenetic signal

• Amplification and sequencing protocols:

  • Design primers for universal amplification across diverse plant lineages

  • For atpH and complementary markers, specific primers with high success rates include:

    • atpH-f: 5'-AACAAAAGGATTCGCAAATAAAAG-3'

    • atpH-r: Not specified in the results but paired with atpH-f

    • atpH-atpI region: atpH-f (5'-AACAAAAGGATTCGCAAATAAAAG-3') and atpI-r (5'-AGTTGTTGTTCTTGTTTCTTTAGT-3')

• Methodological approach for multi-marker phylogenetic analysis:

  • Extract total DNA from plant tissue using CTAB or commercial plant DNA extraction kits

  • Amplify multiple markers using high-fidelity PCR with the Phusion Polymerase

  • Purify PCR products with PEG8000 for direct sequencing

  • Sequence quality assessment: Minimum 600 bp read length with quality values >90% (QV >20)

  • Perform multiple sequence alignment followed by phylogenetic reconstruction using maximum likelihood, Bayesian inference, or other appropriate methods

• Marker selection considerations:

  • Amplification success rate: Choose markers with high amplification success across diverse lineages

  • Sequence quality: Select markers that consistently produce high-quality sequencing reads (>600 bp with QV >20)

  • Variability level: Match marker variability to the taxonomic level being investigated

  • Combined analysis: Employ concatenated datasets of multiple markers for increased phylogenetic resolution

The combination of atpH with other chloroplast markers provides robust phylogenetic inference, with different markers contributing complementary evolutionary signals. The atpH-atpI region shows 100% amplification success rate across diverse plant lineages and produces high-quality sequences (98.1-99.5% quality values), making it particularly valuable in phylogenetic studies .

How can researchers optimize the cleavage of fusion tags from recombinant ATP synthase subunit c?

Optimizing the cleavage of fusion tags from recombinant ATP synthase subunit c requires careful consideration of multiple factors to achieve efficient separation while maintaining protein stability and activity:

• Protease selection based on fusion system:

  • Factor Xa for MBP fusions (pMAL-c2x system): Recognizes IEGR↓X sequence

  • TEV protease for His-tagged proteins: Recognizes ENLYFQ↓G/S sequence

  • Thrombin for GST fusions: Recognizes LVPR↓GS sequence

  • PreScission protease: Offers high specificity with minimal non-specific cleavage

• Critical parameters for optimization:

  • Temperature: Screen 4°C, 16°C, and 25°C; lower temperatures often reduce non-specific cleavage

  • Incubation time: Test time points from 2-24 hours to balance complete cleavage against potential degradation

  • Enzyme:substrate ratio: Typically start with 1:50 to 1:100 (w/w) and adjust based on efficiency

  • Buffer composition: Optimize buffer components including:

    • pH (typically 7.0-8.5 depending on the protease)

    • Salt concentration (50-200 mM NaCl)

    • Required cofactors (e.g., 1-2 mM CaCl₂ for Factor Xa)

• Detergent considerations for membrane proteins:

  • Select detergents compatible with the protease activity

  • Maintain detergent concentration above CMC but below levels that might inhibit protease

  • Common compatible detergents include DDM (0.02-0.1%), LDAO (0.05-0.2%), and OG (0.5-1.0%)

• Monitoring cleavage efficiency:

  • Time-course sampling and analysis by SDS-PAGE

  • Western blotting with antibodies against both the target protein and fusion tag

  • Mass spectrometry to confirm complete removal and precise cleavage site

• Troubleshooting common problems:

  • Insufficient cleavage: Increase enzyme:substrate ratio or incubation time

  • Non-specific cleavage: Reduce temperature, incubation time, or try alternative protease

  • Protein precipitation: Adjust buffer conditions or include stabilizing agents

  • Protease inhibition by detergents: Test different detergent types or concentrations

• Post-cleavage purification strategy:

  • Reverse affinity chromatography to remove uncleaved fusion protein and the cleaved tag

  • Size exclusion chromatography to separate the target protein from protease

  • Buffer exchange to conditions optimal for downstream applications

These methodologies should be adapted for the specific construct and expression system being used. For the pMAL-c2x-malE/atpH system, Factor Xa cleavage can be optimized with these parameters as a starting point, with systematic variation to determine optimal conditions for the specific ATP synthase subunit c protein .

How can comparative analysis of c-ring stoichiometry across species inform synthetic biology applications?

Comparative analysis of c-ring stoichiometry across species provides crucial insights for synthetic biology applications, particularly in designing artificial energy-transducing systems with customized efficiency parameters:

• Methodology for comparative c-ring analysis:

  • Structural determination using cryo-electron microscopy and X-ray crystallography

  • Biochemical approaches including crosslinking and mass spectrometry

  • Sequence analysis of interface residues that determine packing preferences

  • Bioinformatic prediction of stoichiometry based on key sequence features

• Evolutionary patterns and energetic implications:

  • Chloroplastic ATP synthases typically have larger c-rings (14 subunits in spinach) compared to mitochondrial ATP synthases (8-10 subunits)

  • The H⁺/ATP ratio directly correlates with c-ring size (n/3, where n is the number of c-subunits)

  • This ratio reflects adaptation to different proton motive force magnitudes available in different cellular compartments

• Synthetic biology design principles:

  • Engineering c-subunit interface residues to create rings with predetermined stoichiometry

  • Custom-designed c-rings with specific H⁺/ATP ratios for optimized energy conversion in artificial systems

  • Integration of designed ATP synthases into synthetic membranes for bioenergetic applications

• Strategic research approach:

  • Identify key residues controlling oligomerization through conservation analysis

  • Highly conserved interface residues between APP and APLP2 proteins suggest functionally important interaction sites that could be applied to ATP synthase design

  • Create libraries of c-subunit variants with modified interface residues

  • Screen for altered stoichiometry using analytical techniques like SEC-MALS and native mass spectrometry

  • Validate energy conversion efficiency in reconstituted systems

• Applications in synthetic biology:

  • Biohybrid energy conversion devices with tunable efficiency

  • Customized ATP production systems for cell-free biotechnology

  • Artificial photosynthetic systems with optimized energy capture and conversion

  • Biocompatible power sources for nanomachines and biosensors

The systematic comparison of c-ring stoichiometry across species, combined with detailed structural and functional analysis, provides a knowledge base for rational design of artificial ATP synthases with customized properties. The recombinant expression and purification methods developed for ATP synthase subunit c enable the production of engineered variants for these synthetic biology applications .

What technical advances could improve structural characterization of recombinant ATP synthase components?

Several technical advances could significantly enhance structural characterization of recombinant ATP synthase components, addressing current limitations and enabling more detailed analyses:

• Advanced crystallization approaches:

  • Lipidic cubic phase (LCP) crystallization: This method provides a native-like membrane environment that can stabilize membrane proteins like ATP synthase subunit c

  • Crystallization chaperones: Engineered binding proteins (nanobodies, DARPins) that can stabilize specific conformations and provide crystal contacts

  • Microfluidic crystallization platforms: Allow higher-throughput screening with minimal protein consumption

  • Automated crystal harvesting and mounting: Reduces crystal damage during handling

• Cryo-electron microscopy innovations:

  • Direct electron detectors with improved sensitivity and decreased noise

  • Advanced motion correction algorithms for better image processing

  • Phase plates for enhanced contrast of small membrane proteins

  • Focused ion beam milling for in situ structural studies of membrane proteins

  • Time-resolved cryo-EM for capturing different functional states

• Integrative structural biology approaches:

  • Combining X-ray crystallography, cryo-EM, and NMR data into unified structural models

  • Cross-linking mass spectrometry (XL-MS) to map protein-protein interactions

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe dynamics and conformational changes

  • Small-angle X-ray scattering (SAXS) for solution structure determination

• Advanced biophysical characterization:

  • Single-molecule FRET to monitor conformational changes during function

  • High-speed atomic force microscopy (HS-AFM) for visualizing dynamics at the nanoscale

  • Solid-state NMR specifically adapted for membrane proteins

  • Native mass spectrometry for determining intact complex stoichiometry and composition

• Computational approaches:

  • Molecular dynamics simulations to model protein-lipid interactions and conformational dynamics

  • Machine learning algorithms for improved image processing in cryo-EM

  • Integrative modeling platforms that combine data from multiple experimental techniques

  • Quantum mechanics/molecular mechanics (QM/MM) calculations for proton transfer mechanisms

Implementation strategy for structural studies of ATP synthase components:

• Begin with recombinant protein production using optimized systems like pMAL-c2x-malE/atpH

• Perform preliminary characterization using biochemical and biophysical methods

• Apply complementary structural techniques (X-ray, cryo-EM, spectroscopy)

• Integrate data using computational approaches

• Validate structural models through mutagenesis and functional studies

These technical advances would address current challenges in structural biology of ATP synthase components, including difficulty obtaining well-diffracting crystals, challenges in capturing different functional states, and limitations in resolving high-resolution structures of small membrane proteins like ATP synthase subunit c .

How might site-directed mutagenesis of recombinant ATP synthase subunit c inform the design of novel bioenergetic systems?

Site-directed mutagenesis of recombinant ATP synthase subunit c provides powerful insights for designing novel bioenergetic systems with customized properties for biotechnological applications:

• Strategic targeting of functional residues:

  • Proton-binding site: Mutations of the conserved carboxylate residue (Asp or Glu) in the c-subunit can alter:

    • pKa values, potentially changing the pH range for optimal function

    • Proton affinity, modifying the energy threshold for rotation

    • Specificity, potentially allowing transport of ions other than protons

  • Interface residues: Mutations at c-c subunit interfaces can modify:

    • Ring stability, affecting the durability of synthetic systems

    • Assembly efficiency, important for recombinant production

    • Stoichiometry, directly impacting the H⁺/ATP ratio and energy conversion efficiency

• Experimental workflow for structure-function analysis:

  • Design rational mutations based on sequence conservation analysis and structural data

  • Create mutant constructs using site-directed mutagenesis of the atpH gene

  • Express wild-type and mutant proteins using the optimized pMAL-c2x system

  • Purify and reconstitute into liposomes or nanodiscs for functional studies

  • Compare structural stability, assembly properties, and functional parameters

• Applications in designing novel bioenergetic systems:

  • pH-adapted ATP synthases: Mutants with altered pKa values of the proton-binding site could function optimally in non-physiological pH environments

  • Ion-selective variants: Mutations creating specificity for Na⁺ or K⁺ instead of H⁺ would enable new types of ion-gradient-powered systems

  • Efficiency-tuned ATP generators: Altered c-ring stoichiometry through interface mutations could create systems with customized energy conversion ratios

  • Redox-coupled systems: Integration of artificial redox sensors into the c-subunit could create light or electron-driven ATP production systems

• Advanced modification approaches:

  • Incorporation of unnatural amino acids with novel properties

  • Creation of chimeric c-subunits combining features from different species

  • Introduction of synthetic cofactors for enhanced or novel functionality

  • Computational design of completely novel c-subunit variants with predicted properties

• Validation and characterization methods:

  • Structural analysis using CD spectroscopy to confirm proper folding

  • Assembly assessment using analytical ultracentrifugation and SEC-MALS

  • Functional characterization through proton pumping assays in reconstituted systems

  • ATP synthesis measurement in complete or partial ATP synthase systems

The insights gained from such studies can directly inform the design of artificial ATP-generating systems for applications including biohybrid devices, synthetic cells, and biocompatible power sources for nanotechnology. The optimized recombinant expression system for ATP synthase subunit c provides the foundation for this mutational analysis and subsequent synthetic biology applications .