Recombinant Acorus calamus ATP synthase subunit b, chloroplastic (atpF)

What is Recombinant Acorus calamus ATP synthase subunit b, chloroplastic (atpF)?

Recombinant Acorus calamus ATP synthase subunit b (atpF) is a full-length protein derived from the chloroplasts of Acorus calamus (Sweet flag). This protein is part of the ATP synthase complex, specifically localized in the F₀ sector which is embedded in the chloroplast membrane. The recombinant form is produced in vitro using E. coli expression systems with an N-terminal 10xHis-tag to facilitate purification and downstream applications . The protein consists of 184 amino acids and plays a crucial role in energy transduction within chloroplasts, similar to how its mitochondrial counterpart functions in cellular respiration.

How does chloroplastic ATP synthase compare to mitochondrial ATP synthase?

Chloroplastic and mitochondrial ATP synthases share fundamental structural and functional principles but exhibit several key differences:

While mitochondrial ATP synthase uses the proton gradient established by the electron transport chain during cellular respiration, chloroplastic ATP synthase utilizes the proton gradient generated during photosynthesis . Both employ the same basic rotary mechanism where proton translocation through the F₀ sector drives rotation of the central stalk, causing conformational changes in the F₁ sector that catalyze ATP synthesis.

What are the optimal conditions for expression and purification of recombinant Acorus calamus ATP synthase subunit b?

Optimal expression and purification of recombinant Acorus calamus ATP synthase subunit b requires careful consideration of several parameters:

Expression System:

• E. coli is the preferred expression system for this protein, as indicated in the product specifications

• BL21(DE3) or similar strains are typically used for membrane protein expression

• Expression should be conducted at lower temperatures (16-20°C) to enhance proper folding

• IPTG concentration should be optimized (typically 0.1-0.5 mM) to prevent formation of inclusion bodies

Purification Strategy:

• Cell lysis: Use gentle methods such as enzymatic lysis or mild detergents to preserve protein structure

• Solubilization: Employ appropriate detergents (DDM, LDAO, or C12E8) to extract the membrane protein

• Affinity chromatography: Utilize the N-terminal 10xHis-tag with Ni-NTA columns

• Size exclusion chromatography: Further purify the protein and assess oligomeric state

• Buffer optimization: Maintain pH 7.5-8.0 with Tris/PBS-based buffer containing 6% trehalose as used in the commercial preparation

For storage, the protein can be maintained as a liquid at -20°C/-80°C for up to 6 months or as a lyophilized powder for up to 12 months. Repeated freeze-thaw cycles should be avoided to prevent denaturation .

How can researchers assess the functional integrity of purified recombinant ATP synthase subunit b?

Assessing the functional integrity of purified recombinant ATP synthase subunit b requires multiple complementary approaches:

Structural Assessment:

• Circular dichroism (CD) spectroscopy to analyze secondary structure elements

• Intrinsic fluorescence measurements to evaluate tertiary structure integrity

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine oligomeric state

• Limited proteolysis to probe for properly folded domains resistant to digestion

Functional Assessment:

• Binding assays with other ATP synthase subunits, particularly those that interact with subunit b in the native complex

• Reconstitution experiments with other purified subunits to assess assembly capability

• ATP hydrolysis assays using reconstituted complexes in proteoliposomes

• Proton translocation measurements in reconstituted systems

Interaction Validation:

• Pull-down assays using the His-tag to identify binding partners

• Surface plasmon resonance (SPR) to quantify binding affinities with other subunits

• Isothermal titration calorimetry (ITC) to determine thermodynamic parameters of interactions

• Crosslinking experiments to capture transient interactions

These methodologies collectively provide a comprehensive assessment of whether the recombinant protein maintains structural and functional properties comparable to the native form.

What methods are recommended for studying ATP synthase subunit b integration into functional complexes?

Studying the integration of ATP synthase subunit b into functional complexes requires specialized techniques that address both assembly processes and functional outcomes:

Assembly Analysis:

• Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) to visualize intact complexes and subcomplexes

• Two-dimensional BN/SDS-PAGE to identify components of different assembly intermediates

• Sucrose gradient ultracentrifugation to separate complexes based on size

• Chemical crosslinking followed by mass spectrometry to map protein-protein interactions

• Cryo-electron microscopy to visualize the structure of assembled complexes

Functional Reconstitution:

• Co-expression systems incorporating multiple subunits

• Step-wise reconstitution protocols mimicking the proposed assembly pathway where the c-ring is formed first, followed by F₁ binding, stator arm incorporation, and finally addition of subunits a and A6L

• Proteoliposome reconstitution to evaluate proton pumping and ATP synthesis activities

• Patch-clamp studies to measure proton conductance in reconstituted systems

In Vivo Approaches:

• Complementation assays in ATP synthase-deficient bacterial or yeast strains

• Fluorescence microscopy with tagged subunits to track assembly in living cells

• Inducible expression systems to study temporal aspects of complex assembly

These methods align with current understanding of ATP synthase assembly pathways, which involve the formation of separate modules that converge at the final assembly stage .

What role does ATP synthase subunit b play in the dimerization and oligomerization of ATP synthase complexes?

ATP synthase subunit b contributes significantly to the dimerization and oligomerization of ATP synthase complexes, which have important structural and functional implications:

ATP synthase exists not only as monomers but also as dimers and higher-order oligomers, particularly in the mitochondria and chloroplasts. While subunit a forms the primary basis for dimerization due to its multiple transmembrane helices, subunit b, as part of the peripheral stalk, plays a crucial supporting role in stabilizing these higher-order structures .

Specific Roles of Subunit b in Oligomerization:

• Serves as a structural component of the peripheral stalk that stabilizes the monomer-monomer interface

• Interacts with other accessory subunits (e, g, and A6L) to maintain dimer stability

• Contributes to the proper alignment of monomers within the membrane

• Influences the angle between monomers, which affects membrane curvature

Functional Significance:

• Dimerization and oligomerization create local membrane curvature that enhances the efficiency of ATP synthesis

• Higher-order structures stabilize the ATP synthase complex during rapid catalysis

• Organized arrays of ATP synthase dimers can form along membrane ridges, potentially contributing to cristae formation in mitochondria

Methodological Approaches to Study Oligomerization:

• BN-PAGE with varying detergent concentrations to preserve different oligomeric states

• Atomic force microscopy (AFM) to visualize oligomeric arrangements in membranes

• Förster resonance energy transfer (FRET) between labeled subunits to detect dimerization

• Cryo-electron tomography to observe native arrangements in membrane fragments

• Site-directed mutagenesis of specific residues in subunit b to identify dimerization domains

Understanding the role of subunit b in ATP synthase oligomerization contributes to our knowledge of both the structural organization and functional efficiency of these essential energy-transducing complexes .

How can researchers investigate the potential relationship between Acorus calamus ATP synthase components and the plant's reported anti-cancer properties?

Investigating the potential relationship between Acorus calamus ATP synthase components and the plant's reported anti-cancer properties requires multidisciplinary approaches bridging biochemistry, molecular biology, and pharmacology:

Acorus calamus has demonstrated anti-tumor and chemo-preventive activities in numerous pre-clinical studies, with its bioactive components (primarily α- and β-asarone) being the focus of most research . The potential connection between ATP synthase components and these anti-cancer properties represents an unexplored but promising research direction.

Investigative Approaches:

• Isolation and Bioactivity Screening:

  • Fractionate A. calamus extracts to isolate ATP synthase components

  • Screen fractions for cytotoxicity against cancer cell lines

  • Compare activity profiles with known bioactive components (asarones)

• Target Identification Studies:

  • Use labeled recombinant ATP synthase subunit b to identify binding partners in cancer cells

  • Perform competitive binding assays with asarones to determine if they target the same proteins

  • Employ proteomics approaches to identify changes in protein-protein interactions following treatment

• Functional Studies:

  • Assess effects of A. calamus extracts and isolated components on mitochondrial and cellular ATP levels

  • Measure changes in mitochondrial membrane potential and ATP synthase activity

  • Investigate impacts on ATP-dependent processes crucial for cancer cell survival and proliferation

• Mechanistic Investigations:

  • Examine whether A. calamus components affect ATP synthase oligomerization

  • Investigate potential disruption of ATP synthase assembly pathways

  • Explore effects on mitochondrial morphology and function

Experimental Design Table:

This research direction could potentially reveal novel mechanisms underlying the anti-cancer properties of A. calamus beyond the currently studied asarones .

What technical challenges exist in studying the assembly process of ATP synthase incorporating recombinant Acorus calamus subunit b?

Studying ATP synthase assembly with recombinant Acorus calamus subunit b presents several technical challenges that researchers must address:

Expression and Stability Challenges:

• Maintaining proper folding of hydrophobic membrane segments during recombinant expression

• Preserving native-like structure in detergent-solubilized states

• Preventing aggregation during concentration and purification steps

• Ensuring proper post-translational modifications that might be species-specific

Assembly Process Challenges:

• Recreating the stepwise assembly pathway observed in vivo

• Identifying appropriate chaperones needed for correct assembly (such as ATP11 and ATP12, which shield hydrophobic surfaces of unassembled α and β subunits)

• Distinguishing between authentic assembly intermediates and breakdown products that appear under experimental conditions

• Coordinating the addition of mitochondrial/chloroplast-encoded and nuclear-encoded subunits

Methodological Limitations:

• Resolving transient assembly intermediates before they progress to subsequent states

• Developing appropriate in vitro systems that mimic the native membrane environment

• Accounting for the role of membrane potential and pH gradients in assembly

• Translating findings from heterologous expression systems to native chloroplast environments

Proposed Solutions:

• Utilize mild solubilization conditions and amphipathic polymers (e.g., SMALPs) to extract membrane protein complexes

• Employ cell-free expression systems with chloroplast extracts to provide native assembly factors

• Develop fluorescence-based assays to monitor assembly in real-time

• Integrate complementary structural techniques (cryo-EM, crosslinking mass spectrometry) to validate assembly models

• Compare assembly pathways with those established in model systems such as yeast, where ATP synthase forms from three different modules: the c-ring, F₁, and the Atp6p/Atp8p complex

Understanding these challenges and implementing appropriate methodological approaches is crucial for accurate characterization of ATP synthase assembly incorporating recombinant Acorus calamus subunit b.

What emerging technologies could advance our understanding of chloroplastic ATP synthase structure and function?

Several cutting-edge technologies show promise for advancing our understanding of chloroplastic ATP synthase structure and function:

Advanced Structural Biology Approaches:

• Cryo-electron microscopy (cryo-EM) with improved resolution for membrane proteins

• Integrative structural biology combining multiple techniques (X-ray crystallography, NMR, SAXS)

• Time-resolved structural methods to capture dynamic conformational changes during catalysis

• Native mass spectrometry to analyze intact complexes and their stoichiometry

Single-Molecule Techniques:

• High-speed atomic force microscopy to visualize ATP synthase dynamics in real-time

• Single-molecule FRET to monitor conformational changes during rotation

• Optical and magnetic tweezers to measure forces generated during ATP synthesis

• Nanopore-based approaches to study individual ATP synthase complexes

Advanced Genomic and Systems Biology Approaches:

• CRISPR-Cas9 gene editing to create precise modifications in ATP synthase subunits

• Synthetic biology approaches to engineer ATP synthases with novel properties

• Multi-omics integration to understand regulation of ATP synthase in different physiological states

• Computational modeling and simulation of the complete ATP synthase complex

Novel Functional Approaches:

• Optogenetic control of proton gradients to manipulate ATP synthase activity

• Microfluidic platforms to study ATP synthase under controlled environmental conditions

• Biosensor development for real-time monitoring of ATP synthase activity in vivo

• 3D bioprinting of artificial membranes containing reconstituted ATP synthase

These emerging technologies promise to overcome current limitations in our understanding of chloroplastic ATP synthase structure, dynamics, and function, potentially revealing unique aspects of the Acorus calamus ATP synthase that could be exploited for biotechnological or therapeutic applications.

How might comparative studies between Acorus calamus ATP synthase and other plant species inform evolutionary understanding?

Comparative studies between Acorus calamus ATP synthase and those from other plant species offer valuable insights into evolutionary relationships and functional adaptations:

Acorus calamus occupies a unique phylogenetic position as one of the most basal monocotyledonous plants, often referred to as a "living fossil." Its ATP synthase components may therefore preserve ancestral features that have been modified in more recently evolved plant lineages.

Comparative Research Approaches:

• Sequence and Structure Analysis:

  • Phylogenetic analysis of ATP synthase subunit b sequences across plant lineages

  • Identification of conserved domains versus rapidly evolving regions

  • Structural modeling to predict evolutionary constraints on protein folding

  • Analysis of selection pressures on different functional domains

• Functional Comparisons:

  • Comparative biochemical characterization of ATP synthase activity parameters

  • Adaptation to different environmental conditions (temperature, pH, salt tolerance)

  • Regulatory mechanisms controlling ATP synthase assembly and activity

  • Energy coupling efficiency across evolutionary distant species

• Methodological Framework:

  • Recombinant expression of ATP synthase subunits from multiple species

  • Creation of chimeric proteins to identify functionally important domains

  • Complementation studies in heterologous systems

  • Molecular dynamics simulations to identify evolutionarily conserved motion patterns

Evolutionary Insights Table:

These comparative studies could reveal how ATP synthase has evolved to support diverse photosynthetic strategies across plant lineages while maintaining its fundamental role in energy transduction, potentially uncovering unique adaptations in Acorus calamus that contribute to its medicinal properties.

What are the optimal storage and handling protocols for maintaining recombinant Acorus calamus ATP synthase subunit b activity?

Maintaining the activity of recombinant Acorus calamus ATP synthase subunit b requires careful attention to storage and handling protocols:

Storage Conditions:

• Short-term storage (1-2 weeks): 4°C in appropriate buffer with protease inhibitors

• Medium-term storage (1-6 months): -20°C in liquid form with 25-50% glycerol

• Long-term storage (6-12 months): -80°C as either lyophilized powder or aliquoted solution

• The commercial preparation recommends storage at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid freeze-thaw cycles

Buffer Considerations:

• pH maintenance between 7.5-8.0 using Tris/PBS-based buffers

• Addition of 6% trehalose as a stabilizing agent to prevent denaturation during freeze-thaw cycles

• Inclusion of appropriate detergents at concentrations above their critical micelle concentration to maintain solubility of membrane-associated regions

• Addition of reducing agents (DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues

Handling Recommendations:

• Thaw frozen aliquots rapidly in a water bath set to 25°C

• Keep samples on ice during experimental procedures

• Avoid vigorous vortexing; instead, mix by gentle inversion or flicking

• Use low-protein-binding tubes and pipette tips to prevent adsorptive losses

• Monitor protein concentration regularly as membrane proteins may precipitate over time

• When using for functional studies, equilibrate to room temperature 15-30 minutes before assaying

Quality Control Measures:

• Periodically assess protein integrity by SDS-PAGE

• Verify functional activity using appropriate binding or activity assays

• Monitor secondary structure stability using circular dichroism spectroscopy

• Document the number of freeze-thaw cycles and storage duration for each aliquot

Following these optimized protocols will maximize the shelf life and maintain the structural and functional integrity of recombinant Acorus calamus ATP synthase subunit b for extended research applications .

How should researchers troubleshoot common problems encountered when working with ATP synthase components?

Researchers frequently encounter specific challenges when working with ATP synthase components. Here are methodological approaches to troubleshoot common problems:

Problem 1: Low Expression Yields

• Diagnostic Signs: Minimal protein band on SDS-PAGE, weak signal on Western blot

• Troubleshooting Approach:

  • Optimize codon usage for expression host

  • Lower induction temperature (16-18°C)

  • Decrease IPTG concentration (0.1-0.2 mM)

  • Test different E. coli strains (C41/C43 designed for membrane proteins)

  • Co-express with molecular chaperones (GroEL/GroES)

  • Consider using ATP11/ATP12 chaperones which specifically bind to unassembled α and β subunits

Problem 2: Protein Aggregation

• Diagnostic Signs: Precipitation during purification, protein in pellet fraction, high molecular weight bands on native gels

• Troubleshooting Approach:

  • Optimize detergent type and concentration

  • Include stabilizing agents (glycerol, trehalose)

  • Avoid concentrating above critical thresholds

  • Maintain ionic strength above 100 mM

  • Consider using amphipathic polymers instead of detergents

  • Perform size exclusion chromatography immediately after affinity purification

Problem 3: Loss of Activity

• Diagnostic Signs: Purified protein fails to incorporate into functional complexes, no measurable activity

• Troubleshooting Approach:

  • Verify proper folding using circular dichroism

  • Test multiple buffer conditions using thermal stability assays

  • Add lipids representative of native membrane environment

  • Ensure proper redox conditions

  • Consider using gentler purification approaches

  • Verify the presence of required co-factors

Problem 4: Inconsistent Assembly

• Diagnostic Signs: Variable complex formation, incomplete assembly intermediates

• Troubleshooting Approach:

  • Adjust stoichiometric ratios of subunits

  • Include assembly factors from native systems

  • Test stepwise versus simultaneous assembly protocols

  • Optimize membrane/detergent/protein ratios in reconstitution

  • Consider the role of the proton gradient in assembly

  • Follow established assembly pathways (c-ring formation followed by binding of F₁, the stator arm, and finally subunits a and A6L)

Methodological Decision Tree:

These systematic troubleshooting approaches address the specific challenges associated with ATP synthase components while providing methodological solutions based on established research practices.