Recombinant Arabis hirsuta ATP synthase subunit c, chloroplastic (atpH)

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

ATP synthase subunit c forms a ring structure embedded in the thylakoid membrane of chloroplasts. The synthesis of ATP is mechanically coupled to the rotation of this c-subunit ring, which is driven by proton translocation across the membrane along an electrochemical gradient. The c-subunit ring functions as a proton-conducting rotor that converts the energy of proton movement into mechanical rotation, which is then coupled to the catalytic synthesis of ATP in the F₁ region of the enzyme complex .

How does the c-ring stoichiometry affect ATP synthesis efficiency?

The ratio of protons translocated to ATP synthesized varies according to the number of c-subunits (n) per oligomeric ring (c₍ₙ₎). This stoichiometric variation is organism-dependent and directly impacts the bioenergetic efficiency of the ATP synthase. Each c-subunit contains a proton-binding site, so a c-ring with more subunits requires more protons to complete one rotation, resulting in a higher proton-to-ATP ratio. This ratio is inherently related to the metabolic requirements of the organism, though the exact evolutionary factors driving c-ring stoichiometric variation remain incompletely understood .

What is the amino acid sequence of the Arabis hirsuta ATP synthase subunit c (atpH)?

While the search results don't provide the specific sequence for Arabis hirsuta atpH, they do provide information about related proteins. The atpI subunit from Arabis hirsuta has the amino acid sequence: MNVLSCSINTLIKEGLYEISGVEVGQHFYWQIGGFQVHAQVLITSWVVIAILLGSAVLAVRNPQTIPTDGQNFFEFVLEFIRDVSQTQIGEEYGPWVPFIGTLFLFIFVSNWSGALLPWKIIQLPQGELAAPTNDINTTVALALLTSAAYFYAGLSKKGLGYFSKYIQPTPILLPINILEDFTKPLSLSFRLFGNILADELVVVVLVSLVPLVVPIPVMFLGLFTSGIQALIFATLAAAYIGESMEGHH . The atpH gene sequence would need to be determined through genomic analysis or using information from closely related Brassicaceae species.

What are the optimal expression systems for recombinant production of chloroplastic ATP synthase subunit c?

For the recombinant expression of chloroplastic ATP synthase subunit c, E. coli BL21 derivative cells have been successfully used. Due to the hydrophobic nature of the c-subunit, direct expression often leads to inclusion body formation. A proven approach is to express the protein as a fusion with maltose binding protein (MBP), which increases solubility. The optimal expression system uses a plasmid with a codon-optimized gene insert where the hydrophobic c₁ subunit is first expressed as a soluble MBP-c₁ fusion protein. For Arabis hirsuta specifically, researchers should consider using a similar system to that developed for spinach (Spinacia oleracea) chloroplast ATP synthase subunit c .

What purification strategy is most effective for isolating recombinant ATP synthase subunit c?

The most effective purification strategy involves:

• Expression of the c-subunit as an MBP fusion protein to enhance solubility

• Cell lysis under conditions that maintain protein solubility (often using buffers containing 20 mM Tris-HCl pH 8.0 with protease inhibitors)

• Cleavage of the fusion protein to release the c-subunit

• Purification of the cleaved c-subunit using reversed-phase chromatography

This approach has been demonstrated to yield significant quantities of highly purified c₁ subunit with confirmed alpha-helical secondary structure, which is essential for functional studies .

How can researchers verify the correct folding and structure of purified recombinant ATP synthase subunit c?

To verify the correct folding and structure of purified recombinant ATP synthase subunit c, researchers should:

• Perform circular dichroism (CD) spectroscopy to confirm the alpha-helical secondary structure characteristic of ATP synthase subunit c

• Use size-exclusion chromatography to assess the oligomeric state of the protein

• Employ mass spectrometry to confirm protein identity and detect any post-translational modifications

• Conduct structural analysis through techniques such as NMR spectroscopy or X-ray crystallography for detailed structural information

Additionally, functional assays measuring proton translocation activity or reconstitution into liposomes can provide evidence of proper folding .

How conserved is the atpH gene across different Brassicaceae species?

The atpH gene is highly conserved across Brassicaceae species, reflecting its essential role in ATP synthesis. Comparative genomic analyses of chloroplast genomes in the Brassicaceae family show that gene content and organization are generally conserved, with only minor divergences in protein-coding regions. Unlike some other chloroplast genes (such as rps16, infA, or ycf15) that show pseudogenization or complete loss in certain lineages, atpH tends to be well-preserved due to its critical function in the ATP synthase complex .

What evolutionary patterns are observed in ATP synthase c-subunits across different plant species?

ATP synthase c-subunits show interesting evolutionary patterns across plant species, particularly in terms of stoichiometric variation in the c-ring structure. While the basic function remains conserved, the number of c-subunits in the ring varies between species, affecting the proton-to-ATP ratio. This variation is thought to be an adaptive response to different metabolic demands and environmental conditions. Comparative studies of synonymous (KS) and nonsynonymous (KA) substitution rates in chloroplast genes, including those encoding ATP synthase components, help identify regions under selective pressure .

How do mutations in the atpH gene affect ATP synthase function and plant fitness?

• Altering the proton-binding sites in the c-subunit, affecting proton translocation efficiency

• Disrupting the proper assembly of the c-ring structure

• Changing the protein-protein interactions between the c-subunit and other components of the ATP synthase complex

• Potentially affecting the stoichiometry of the c-ring, altering the proton-to-ATP ratio

These effects can compromise ATP production efficiency, reducing energy availability for photosynthetic metabolism and other cellular processes, ultimately affecting plant growth and adaptation to environmental conditions .

How can site-directed mutagenesis of atpH be used to investigate c-ring assembly and function?

Site-directed mutagenesis of atpH can be a powerful tool for investigating c-ring assembly and function by:

• Creating targeted mutations in amino acids involved in proton binding to analyze their impact on proton translocation

• Modifying residues at the interfaces between c-subunits to study their role in c-ring assembly

• Introducing mutations that alter the interactions between the c-ring and other ATP synthase components

• Engineering changes that might affect the stoichiometry of the c-ring to study the relationship between structure and function

For example, researchers could modify conserved residues in the transmembrane helices that are involved in c-subunit interactions to determine their role in c-ring stability and assembly .

What techniques are available for studying the dynamics of c-ring rotation in ATP synthase?

Advanced techniques for studying c-ring rotation dynamics include:

• Single-molecule fluorescence resonance energy transfer (FRET) to observe real-time rotational movements

• Optical or magnetic tweezers to measure the torque generated during rotation

• High-speed atomic force microscopy (HS-AFM) to visualize conformational changes

• Molecular dynamics simulations to model rotation at the atomic level

• Cryo-electron microscopy to capture different conformational states of the rotating complex

These techniques can provide insights into the mechanistic details of how proton translocation drives c-ring rotation and how this rotation is coupled to ATP synthesis .

What approaches can be used to reconstitute functional c-rings from recombinant c-subunits?

Approaches for reconstituting functional c-rings from recombinant c-subunits include:

• Detergent-mediated reconstitution, where purified c-subunits are mixed with appropriate detergents that facilitate self-assembly

• Lipid-based reconstitution methods, incorporating c-subunits into liposomes or nanodiscs

• Stepwise assembly with other ATP synthase components to form partial or complete complexes

• Co-expression systems where multiple ATP synthase subunits are expressed simultaneously

• Cell-free protein synthesis systems coupled with membrane-like environments

Successful reconstitution can be verified by:

• Electron microscopy to visualize ring structures

• Proton translocation assays to test functionality

• ATP synthesis assays when incorporated with other ATP synthase components

• Structural studies using cryo-EM or X-ray crystallography to confirm proper assembly

How do environmental factors influence expression and assembly of ATP synthase c-subunits in plants?

Environmental factors influence the expression and assembly of ATP synthase c-subunits through:

• Light intensity effects: High light conditions may upregulate ATP synthase components to meet increased energy demands

• Temperature responses: Both heat and cold stress can alter expression patterns and affect protein folding and assembly

• Nutrient availability: Phosphate limitation can impact ATP synthesis machinery

• Drought stress: Water limitation affects thylakoid membrane integrity and ATP synthase assembly

• Salt stress: Ionic imbalances can disrupt proton gradients essential for ATP synthase function

Research approaches to study these effects include:

• Transcriptomic analysis under various environmental conditions

• Proteomics to quantify protein levels and post-translational modifications

• Electron microscopy to examine structural changes in thylakoid membranes

• Functional assays to measure ATP synthesis capacity

• Genetic approaches using mutants with altered ATP synthase components

What are the main challenges in expressing and purifying membrane proteins like ATP synthase subunits?

The main challenges in expressing and purifying membrane proteins like ATP synthase subunits include:

• Insolubility due to their hydrophobic nature

• Potential toxicity to host cells when overexpressed

• Improper folding in heterologous expression systems

• Difficulties in extracting proteins from membranes

• Maintaining protein stability during purification

• Low expression yields

Effective solutions include:

• Using fusion partners like MBP to enhance solubility

• Optimizing codon usage for the expression host

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

• Careful selection of detergents for extraction and purification

• Developing optimized lysis conditions with appropriate protease inhibitors

• Using column purification methods specifically designed for hydrophobic proteins, such as reversed-phase chromatography

How can researchers optimize codon usage for improved expression of Arabis hirsuta atpH in E. coli?

To optimize codon usage for improved expression of Arabis hirsuta atpH in E. coli:

• Analyze the native atpH sequence for rare codons in E. coli

• Replace rare codons with synonymous codons that are more abundant in E. coli

• Adjust the GC content to match E. coli preferences

• Consider using gene design software like Gene Designer by DNA2.0

• Include appropriate restriction sites for cloning

• Optimize 5' and 3' untranslated regions for efficient translation

The approach used for spinach chloroplast ATP synthase c-subunit, which involved designing a synthetic gene with codons optimized for E. coli expression and terminal restriction sites for cloning, provides a useful model for Arabis hirsuta atpH optimization .

What analytical methods are most suitable for characterizing the oligomeric state of ATP synthase c-rings?

The most suitable analytical methods for characterizing the oligomeric state of ATP synthase c-rings include:

• Blue Native-PAGE: Allows separation of intact protein complexes under non-denaturing conditions

• Size-exclusion chromatography: Separates proteins based on their hydrodynamic radius

• Analytical ultracentrifugation: Provides accurate determination of molecular mass and shape

• Mass spectrometry: Native MS can measure the mass of intact complexes

• Cryo-electron microscopy: Enables direct visualization and counting of c-subunits in the ring

• Atomic force microscopy: Provides topographical information about the c-ring structure

• Chemical cross-linking combined with mass spectrometry: Identifies interacting regions between subunits

These methods can be used complementarily to provide a comprehensive characterization of c-ring stoichiometry and structure .