Recombinant Coffea arabica ATP synthase subunit c, chloroplastic (atpH)

How does the structure of ATP synthase subunit c relate to its function in photosynthesis?

The ATP synthase subunit c adopts a primarily alpha-helical secondary structure, typically consisting of two transmembrane helices connected by a hydrophilic loop . This structure is critical for its function in the c-ring, which acts as a rotor in the FoF1-ATP synthase complex. The c-subunit contains conserved glutamic acid residues that undergo protonation and deprotonation cycles, facilitating proton movement across the thylakoid membrane . These proton binding sites are essential for converting the energy of the proton gradient into mechanical rotation. The number of c-subunits forming the oligomeric ring varies among species and directly influences the bioenergetic efficiency of ATP synthesis, as it determines the ratio of protons translocated to ATP molecules synthesized . This stoichiometric relationship is inherently tied to the metabolism of the organism, though the exact causes of c-ring stoichiometry variation are not fully understood .

What is known about the genetic regulation of atpH expression in Coffea arabica?

The atpH gene in Coffea arabica is located in the chloroplast genome and encodes the ATP synthase subunit c protein. Like most chloroplast genes, its expression is regulated through a combination of transcriptional, post-transcriptional, and translational control mechanisms. Expression levels typically correlate with photosynthetic activity, showing higher expression in mature leaves compared to non-photosynthetic tissues. The regulation involves both nucleus-encoded factors and plastid-specific mechanisms, creating a complex regulatory network that coordinates chloroplast gene expression with nuclear gene expression. Environmental factors such as light intensity, temperature, and water availability can modulate atpH expression as part of adaptive responses in coffee plants. Studying the genetic regulation of atpH requires techniques such as quantitative RT-PCR, RNA-seq analysis of different tissues and developmental stages, and promoter analysis to identify regulatory elements controlling expression patterns.

What are the most effective methods for recombinant expression of the hydrophobic ATP synthase subunit c?

Effective recombinant expression of the hydrophobic Coffea arabica ATP synthase subunit c requires specialized approaches to overcome the challenges associated with membrane proteins. Based on successful methods with other chloroplastic ATP synthase c-subunits, an effective strategy involves expressing the protein as a fusion with a solubility-enhancing partner such as maltose binding protein (MBP) . The recommended procedure includes:

• Gene synthesis with codon optimization for the expression host (typically E. coli)

• Cloning into a vector containing an MBP fusion tag with a cleavable linker

• Expression in BL21 derivative E. coli cells, which have been successful for other eukaryotic c-subunits

• Induction under optimized conditions (temperature, IPTG concentration, duration)

• Purification of the soluble MBP-c1 fusion protein using affinity chromatography

• Controlled proteolytic cleavage to separate the c-subunit from MBP

• Final purification of the cleaved c-subunit using reversed-phase chromatography

This approach has been successfully applied to spinach chloroplast ATP synthase c-subunit and can be adapted for the coffee homolog, enabling soluble expression of this eukaryotic membrane protein in bacterial systems .

How can researchers optimize purification protocols for recombinant ATP synthase subunit c?

Purification of recombinant Coffea arabica ATP synthase subunit c requires specialized approaches due to its hydrophobic nature. A systematic purification strategy, based on successful methods for other chloroplastic c-subunits, involves:

• Initial purification as a fusion protein (e.g., MBP-c1) using affinity chromatography to obtain a soluble preparation

• Proteolytic cleavage of the fusion protein under controlled conditions to release the c-subunit

• Reversed-phase chromatography as a critical step for final purification of the cleaved c-subunit

• Verification of purified protein using SDS-PAGE, mass spectrometry, and Western blotting

The reversed-phase column purification is particularly effective for isolating the highly hydrophobic c-subunit . Purification buffers should be carefully optimized, potentially including mild detergents to maintain protein solubility. For analytical purposes, researchers should confirm that the purified protein has the correct alpha-helical secondary structure using circular dichroism spectroscopy . This verification step ensures that the recombinant protein has folded properly and is suitable for structural and functional studies.

What techniques are recommended for verifying proper folding of recombinant ATP synthase subunit c?

Verifying proper folding of recombinant Coffea arabica ATP synthase subunit c is essential for ensuring functional relevance. Recommended analytical techniques include:

• Circular Dichroism (CD) Spectroscopy: The primary method to confirm the alpha-helical secondary structure characteristic of c-subunits . The CD spectrum should show typical signatures of alpha-helical proteins with negative peaks at 208 and 222 nm.

• Fourier Transform Infrared (FTIR) Spectroscopy: Provides complementary information about secondary structure, particularly useful for membrane proteins.

• Limited Proteolysis: Properly folded proteins show distinct proteolytic patterns compared to misfolded variants.

• Thermal Stability Assays: Differential scanning calorimetry or thermal shift assays can assess protein stability and compare it with c-subunits from other species.

• Functional Reconstitution: Incorporation into liposomes to test proton translocation capability or binding of known inhibitors like DCCD (N,N'-dicyclohexylcarbodiimide).

• Native PAGE or Size Exclusion Chromatography: To assess oligomerization behavior, as properly folded c-subunits should show characteristic oligomerization patterns.

Confirming proper folding is crucial before proceeding to more advanced structural or functional studies, as the hydrophobic nature of the c-subunit makes it prone to misfolding during recombinant expression .

How can researchers investigate c-ring stoichiometry in Coffea arabica ATP synthase?

Investigating c-ring stoichiometry (the number of c-subunits per ring) in Coffea arabica ATP synthase requires specialized approaches:

• Atomic Force Microscopy (AFM):

  • Prepare purified c-rings on atomically flat surfaces

  • Scan individual c-rings to directly count subunits

  • Perform statistical analysis across multiple complexes

• Cryo-Electron Microscopy:

  • Vitrify purified c-rings or intact ATP synthase complexes

  • Collect high-resolution images and perform single-particle analysis

  • Generate 3D reconstructions to determine symmetry and count subunits

• Mass Spectrometry Approaches:

  • Native mass spectrometry of intact c-rings to determine total molecular weight

  • Calculate the number of subunits based on the known mass of monomeric c-subunit

  • Cross-linking coupled with mass spectrometry to identify neighboring subunits

• Functional Proton/ATP Ratio Measurements:

  • Reconstitute ATP synthase in liposomes with pH indicators

  • Measure H+/ATP ratio during catalysis, which correlates with c-ring stoichiometry

  • Compare results with known ratios from other species

The c-ring stoichiometry directly affects the bioenergetic efficiency of ATP synthesis, as it determines the number of protons required to synthesize one ATP molecule . This stoichiometric variation is inherently related to the metabolism of the organism, though the exact causes remain not well understood .

What approaches can be used to study cooperation among c-subunits in Coffea arabica ATP synthase?

Investigating cooperation among c-subunits in Coffea arabica ATP synthase requires sophisticated experimental approaches that build upon techniques demonstrated in other systems. Based on studies with Bacillus PS3 ATP synthase , effective research strategies include:

• Creating a Genetically Fused c-ring:

  • Engineer a single polypeptide containing multiple c-subunits in a defined order

  • This approach allows precise introduction of mutations at specific positions within the c-ring

• Strategic Mutagenesis:

  • Introduce mutations (such as E→D) at varying positions within the fused c-ring

  • Create combinations of single and double mutants at different distances apart

• Functional Assays:

  • Measure ATP synthesis activity in the various mutant constructs

  • Assess ATP-driven proton pumping using fluorescent probes like ACMA

  • Test DCCD-sensitive ATP hydrolysis activity

• Molecular Dynamics Simulations:

  • Model proton transfer events and their coupling across multiple c-subunits

  • Simulate the effects of mutations on proton uptake timing

Studies in Bacillus PS3 have shown that activity decreases further as the distance between mutation sites increases, indicating cooperation among c-subunits . The simulations revealed that prolonged proton uptake in mutated c-subunits can be shared between two c-subunits, explaining the cooperation observed in biochemical assays .

How can molecular simulations be used to study proton transfer in the ATP synthase c-ring?

Molecular simulations provide powerful tools to study proton transfer dynamics in Coffea arabica ATP synthase c-ring at atomic resolution. Effective simulation strategies include:

• Quantum Mechanics/Molecular Mechanics (QM/MM) Simulations:

  • Treat the proton binding site with quantum mechanical methods

  • Model the rest of the protein with classical molecular mechanics

  • Calculate energy barriers for protonation/deprotonation events

• Constant pH Molecular Dynamics:

  • Simulate the protein under varying pH conditions

  • Capture protonation state changes dynamically during the simulation

  • Model the effect of local environment on pKa values of key residues

• Proton Transfer-Coupled Molecular Dynamics:

  • Implement algorithms that model proton hopping through hydrogen bond networks

  • Correlate proton movements with conformational changes in the c-ring

  • Use enhanced sampling techniques to capture long-timescale events

• Multi-Conformation/Molecular Dynamics (MC/MD) Simulations:

  • Model multiple protonation states of c-subunits simultaneously

  • Determine preferred pathways with two or three deprotonated c-subunits

  • Capture cooperative behaviors among neighboring c-subunits

Recent studies using such simulations have revealed that two or three deprotonated carboxyl residues typically face the a-subunit in the wild-type enzyme . Simulations have also shown that the waiting time for proton uptake can be shared between two or three c-subunits, but this sharing decreases as the distance between subunits increases .

What mutations in ATP synthase subunit c are most critical for studying proton translocation?

Key mutations in ATP synthase subunit c for studying proton translocation focus on the conserved glutamic acid residue that serves as the proton binding site. Based on studies in other organisms, particularly informative mutations include:

• Glutamic Acid to Aspartic Acid (E→D):

  • Preserves the carboxyl group but shortens the side chain by one methylene group

  • Partially retains ATP synthesis and proton pump activities

  • Changes the pKa of the proton binding site, affecting protonation/deprotonation kinetics

  • Valuable for studying subtle structural effects on proton transfer

• Glutamic Acid to Glutamine (E→Q):

  • Eliminates the carboxyl group while maintaining side chain length

  • Completely abolishes ATP synthesis activity, ATP-driven proton pump activity, and DCCD-sensitive ATP hydrolysis

  • Demonstrates the absolute requirement for a carboxyl group capable of protonation/deprotonation

• Double E→D Mutations at Varying Distances:

  • Reveals cooperation between c-subunits during rotation

  • Shows greater activity reduction as distance between mutations increases

  • Indicates functional coupling between neighboring subunits

• Conservative Mutations Around the Proton Binding Site:

  • Modify the microenvironment of the key glutamic acid

  • Alter proton affinity without directly changing the binding residue

  • Help map the proton transfer pathway

These mutations provide insights into the mechanism of proton translocation, the cooperation among c-subunits, and the structural features essential for ATP synthase function .

How does the efficiency of ATP synthesis correlate with c-ring structure in different species?

The efficiency of ATP synthesis is directly related to the c-ring structure, particularly its stoichiometry (number of c-subunits per ring), which varies among different species:

*Predicted values; experimental verification required

What are effective protocols for studying ATP synthase inhibitors using recombinant subunit c?

Studying ATP synthase inhibitors using recombinant Coffea arabica subunit c requires systematic approaches:

• Binding Assays with Labeled Inhibitors:

  • Use radioactive or fluorescently labeled DCCD (N,N'-dicyclohexylcarbodiimide)

  • Quantify binding to purified recombinant c-subunit

  • Perform competition assays with other potential inhibitors

• Functional Reconstitution Systems:

  • Incorporate recombinant c-subunit into liposomes or nanodiscs

  • Measure proton translocation using pH-sensitive dyes like ACMA

  • Test inhibitor effects on proton pumping activity

• Thermal Shift Assays:

  • Monitor protein thermal stability in the presence of inhibitors

  • Detect binding by changes in melting temperature

  • Screen multiple compounds in parallel

• Site-Directed Mutagenesis:

  • Create mutations at potential inhibitor binding sites

  • Test for resistance to inhibition

  • Map the binding site through systematic mutation analysis

• Structural Analysis of Inhibitor Complexes:

  • Crystallize or perform NMR on c-subunit with bound inhibitors

  • Determine atomic-level interactions

  • Guide rational design of new inhibitors

This approach provides a platform for identifying novel ATP synthase inhibitors with potential applications in controlling plant pathogens or developing new research tools. The hydrophobic nature of recombinant c-subunit makes it particularly suitable for studying lipophilic inhibitors that target the membrane-embedded Fo sector.

How can we design experiments to study the assembly of c-subunits into functional c-rings?

Designing experiments to study c-subunit assembly into functional c-rings requires multiple complementary approaches:

• In Vitro Assembly Systems:

  • Purify recombinant c-subunits using the MBP-fusion strategy

  • Reconstitute in detergent micelles or lipid bilayers under controlled conditions

  • Monitor oligomerization using native PAGE, analytical ultracentrifugation, or light scattering

• Fluorescence-Based Assays:

  • Label c-subunits with fluorescent tags at non-interfering positions

  • Monitor assembly using Förster resonance energy transfer (FRET)

  • Quantify assembly kinetics and stability in real-time

• Crosslinking Studies:

  • Apply chemical crosslinkers to capture assembly intermediates

  • Analyze products using mass spectrometry to identify interaction interfaces

  • Use variable-length crosslinkers to map spatial relationships

• Co-expression Systems:

  • Co-express c-subunits with other Fo components in bacterial systems

  • Isolate assembled complexes and characterize composition

  • Compare assembly efficiency with mutations affecting key interfaces

• Single-molecule Techniques:

  • Use atomic force microscopy to visualize assembly intermediates

  • Apply force spectroscopy to measure stability of c-ring assemblies

  • Track individual assembly events using total internal reflection fluorescence microscopy

These approaches provide insights into the process of c-ring assembly, which is critical for understanding ATP synthase biogenesis and identifying potential intervention points for modulating ATP synthase function in plants.