Recombinant Acidothermus cellulolyticus ATP synthase subunit b (atpF)
Description
Introduction to Recombinant Acidothermus cellulolyticus ATP Synthase Subunit b (atpF)
Recombinant Acidothermus cellulolyticus ATP synthase subunit b (atpF) is a genetically engineered protein derived from the thermophilic bacterium Acidothermus cellulolyticus. This organism is known for its ability to degrade cellulose, a key component of plant cell walls, making it a valuable source of enzymes for industrial applications, such as biofuel production and bioremediation . The ATP synthase subunit b (atpF) plays a crucial role in the ATP synthesis process, which is essential for energy production in cells.
Background on Acidothermus cellulolyticus
Acidothermus cellulolyticus is a thermophilic actinobacterium that thrives in high-temperature environments. Its genome has been fully sequenced, revealing a diverse array of enzymes capable of degrading plant biomass . The organism's thermophilic nature and its ability to produce enzymes that break down cellulose make it an attractive candidate for biotechnological applications.
Function of ATP Synthase Subunit b (atpF)
ATP synthase is a complex enzyme responsible for generating ATP from ADP and inorganic phosphate using the energy derived from a proton gradient across the cell membrane. The subunit b (atpF) is part of the stalk subunits that connect the membrane-bound F0 sector to the soluble F1 sector of ATP synthase, facilitating the rotation necessary for ATP synthesis .
Recombinant Expression of atpF
Recombinant expression involves producing the atpF subunit in a host organism, typically Escherichia coli, to facilitate large-scale production and purification of the protein. This approach allows for the study of the protein's structure and function in isolation and can be used to develop novel biotechnological tools .
Table 1: Characteristics of Acidothermus cellulolyticus and Its ATP Synthase
| Characteristic | Description |
|---|---|
| Organism | Thermophilic actinobacterium |
| Genome Size | Approximately 2.44 Mb |
| G+C Content | 66.9% |
| ATP Synthase Role | Essential for energy production through ATP synthesis |
| Subunit b (atpF) Function | Connects F0 and F1 sectors, facilitating rotation for ATP synthesis |
Table 2: Potential Applications of Recombinant atpF
| Application | Description |
|---|---|
| Biotechnology | Enzyme production for biofuel and bioremediation |
| Structural Biology | Study of thermophilic adaptations in ATP synthase |
| Energy Research | Understanding efficient energy production mechanisms |
References Barabote, R. D. et al. (2009). Complete genome sequence of the cellulolytic thermophile Acidothermus cellulolyticus 11B. Zhang, Y. et al. (2023). Inhibitors of ATP Synthase as New Antibacterial Candidates. Not directly relevant to the topic. Kumar, R. et al. (2010). Medicinal Chemistry of ATP Synthase: A Potential Drug Target of Dietary Polyphenols and Amphibian Antimicrobial Peptides. Creative Biomart. Recombinant Full Length Roseiflexus Sp. ATP Synthase Subunit B (atpF) Protein. Not directly relevant to the topic. Not directly relevant to the topic. Kumar, P. et al. (2023). An overview of ATP synthase, inhibitors, and their toxicity.
Product Specs
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Target Background
F1F0 ATP synthase synthesizes ATP from ADP using a proton or sodium gradient. This enzyme comprises two domains: the F1 domain, containing the extramembrane catalytic core; and the F0 domain, housing the membrane proton channel. These domains are connected by a central and peripheral stalk. ATP synthesis within the F1 catalytic domain is coupled to proton translocation via a rotary mechanism involving the central stalk subunits. This protein is a component of the F0 channel, forming part of the peripheral stalk which links F1 and F0.
KEGG: ace:Acel_0649
STRING: 351607.Acel_0649
Q&A
What is the structural organization of ATP synthase subunit b in Acidothermus cellulolyticus?
The ATP synthase subunit b in Acidothermus cellulolyticus, like in other bacteria, likely forms a peripheral stalk in the F1F0 ATP synthase complex. Based on structural studies of homologous proteins, the b subunit forms a homodimer with an elongated alpha-helical structure. The dimerization domain typically spans residues 62-122 and creates an extremely elongated structure with a maximal dimension of approximately 95 Å and a radius of gyration of 27 Å . The crystal structure of the dimerization domain in related organisms reveals an isolated, monomeric alpha helix with a length of about 90 Å . The thermophilic nature of Acidothermus cellulolyticus suggests that its atpF product may contain additional stabilizing elements compared to mesophilic counterparts.
How does the b subunit contribute to ATP synthase function in thermophilic bacteria?
In ATP synthase complexes, the b subunit dimer constitutes the peripheral stalk that prevents rotation of the F1 portion during catalysis . The b subunits make unique contributions to the functions of the peripheral stalk, as demonstrated by complementation studies in other systems where two defective b subunits can assemble into a functional F1F0 ATP synthase complex . This suggests each b subunit performs distinct roles in maintaining the structural integrity and function of the complex. In thermophilic organisms like Acidothermus cellulolyticus, the b subunit would need additional stabilizing features to maintain its structural integrity at elevated temperatures, similar to other thermostable proteins from this organism.
What are the key conserved residues in ATP synthase subunit b that are critical for function?
Studies of ATP synthase b subunits have identified several critical residues. An evolutionarily conserved arginine (bArg-36 in E. coli) has been shown to be crucial for F1F0 ATP synthase function . Additionally, the last four C-terminal amino acids of the b subunit play an important role in enzyme assembly . These residues likely mediate interactions with other subunits of the ATP synthase complex. While specific conservation patterns in Acidothermus cellulolyticus atpF have not been detailed in the provided research, these functionally important regions are typically preserved across bacterial species with adaptations that reflect thermal stability requirements.
What expression systems work best for recombinant production of Acidothermus cellulolyticus ATP synthase subunit b?
For recombinant expression of thermostable proteins from Acidothermus cellulolyticus, E. coli expression systems with heat-shock promoters or T7-based expression systems have proven effective. When expressing the atpF gene, consider the following methodological approach:
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Vector selection: pET series vectors with T7 promoters provide high-level expression
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Host strain selection: BL21(DE3) or Rosetta strains accommodate potential codon bias
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Induction conditions: Lower temperatures (18-25°C) despite protein thermostability to ensure proper folding
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Solubility enhancement: Fusion tags such as MBP or SUMO can improve solubility
Given that b subunits tend to form dimers, co-expression strategies similar to those used for studying F1F0 ATP synthase complexes containing two different b subunits could be employed . These systems allow expression of differentially tagged b subunits to study heterodimer formation and function.
What purification strategy should be employed for isolating recombinant Acidothermus cellulolyticus ATP synthase subunit b?
A methodological purification strategy for Acidothermus cellulolyticus ATP synthase subunit b would involve:
| Purification Step | Method | Rationale |
|---|---|---|
| Initial Capture | IMAC (if His-tagged) | Provides high affinity capture of tagged protein |
| Heat Treatment | 60-70°C for 15-20 min | Exploits thermostability to remove host proteins |
| Ion Exchange | Q or SP Sepharose | Removes remaining contaminants based on charge |
| Size Exclusion | Superdex 200 | Separates dimeric b subunit from aggregates/monomers |
The thermostability of proteins from Acidothermus cellulolyticus allows for a heat treatment step that significantly enhances purity, similar to approaches used for other thermostable enzymes from this organism . After purification, confirm identity by mass spectrometry and verify function through ATP hydrolysis assays in reconstituted systems.
How can the oligomeric state of Acidothermus cellulolyticus ATP synthase subunit b be characterized?
The oligomeric state of the ATP synthase subunit b can be characterized using multiple complementary techniques:
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Analytical ultracentrifugation: This technique can determine sedimentation coefficients and frictional ratios, which for b subunit dimers typically show an elongated structure with a frictional ratio around 1.60
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Small-angle X-ray scattering (SAXS): SAXS analysis can provide information on the maximal dimension (typically ~95 Å for b subunit dimers) and radius of gyration (~27 Å)
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Chemical crosslinking: Using bifunctional reagents followed by SDS-PAGE analysis to trap and visualize dimeric species
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Native PAGE: To compare migration patterns with known standards and detect higher-order assemblies
When interpreting results, consider that high temperatures might affect the stability of the dimer, and experimental conditions should be optimized to reflect physiological conditions for thermophilic bacteria.
How does the thermal stability of Acidothermus cellulolyticus ATP synthase subunit b compare to mesophilic homologs?
The thermal stability of proteins from Acidothermus cellulolyticus is typically enhanced compared to mesophilic counterparts, as observed in other enzymes from this organism . This thermostability likely results from multiple structural adaptations:
| Stabilizing Feature | Typical Contribution in Thermophilic Proteins |
|---|---|
| Ion Pairs | Increased number, often in networks |
| Hydrogen Bonds | Higher density, especially in secondary structures |
| Hydrophobic Core | More compact, with increased branched amino acids |
| Surface Residues | Higher proportion of charged residues |
| Proline Content | Increased, particularly in loops |
A directed evolution approach similar to that used for other thermophilic enzymes could be employed to study these features, using random mutagenesis to investigate how altering these elements affects thermal stability . Differential scanning calorimetry (DSC) and circular dichroism (CD) would be effective methods to quantify thermal stability differences between wild-type and mutant forms.
What approaches can be used to study the interaction between ATP synthase subunit b and other components of the F1F0 complex?
To investigate interactions between the b subunit and other components of the ATP synthase complex, consider these methodological approaches:
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Co-immunoprecipitation studies with tagged versions of the b subunit to identify binding partners
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Surface plasmon resonance (SPR) to measure binding kinetics between the b subunit and purified F1 components
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Crosslinking mass spectrometry (XL-MS) to identify specific residues involved in subunit interactions
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Genetic complementation assays similar to those used in E. coli studies, where expression of differentially mutated b subunits can reveal functional interactions
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Cryo-electron microscopy of reconstituted complexes to visualize the structural arrangement
When designing interaction studies, consider that the b subunit makes asymmetric contacts with the F1 portion and that the two b subunits may have distinct interaction profiles despite being identical in sequence .
How can site-directed mutagenesis be used to probe the functional domains of Acidothermus cellulolyticus ATP synthase subunit b?
Site-directed mutagenesis can systematically probe the functional architecture of ATP synthase subunit b through the following methodological approach:
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Target the evolutionarily conserved arginine (homologous to bArg-36 in E. coli) to assess its importance in the thermophilic context
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Modify the C-terminal region (last four amino acids) known to be critical for assembly
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Introduce substitutions in the dimerization domain (residues 62-122) to investigate dimer stability at elevated temperatures
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Create chimeric constructs by swapping domains between thermophilic and mesophilic b subunits to identify regions contributing to thermostability
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Develop a heterodimeric expression system similar to that described for E. coli to express one mutant b subunit with a wild-type version
Functional assessment should include ATP synthesis/hydrolysis assays, assembly analysis by native PAGE, and thermal stability measurements using CD spectroscopy at different temperatures.
What are common issues when working with recombinant thermostable proteins and how can they be addressed?
When working with recombinant thermostable proteins like Acidothermus cellulolyticus ATP synthase subunit b, researchers commonly encounter these challenges:
| Challenge | Solution |
|---|---|
| Low expression levels | Optimize codon usage for expression host; use strong promoters like T7; test multiple growth temperatures |
| Inclusion body formation | Express at lower temperatures (15-25°C); use solubility-enhancing tags (SUMO, MBP); add low concentrations of mild detergents |
| Improper folding | Include molecular chaperones (GroEL/ES) during expression; allow post-purification refolding at elevated temperatures |
| Loss of activity during purification | Include stabilizing agents (glycerol, specific ions); minimize freeze-thaw cycles; store at higher temperatures than mesophilic proteins |
| Oligomerization issues | Optimize buffer conditions (pH, salt concentration); include stabilizing ligands; adjust protein concentration |
For membrane-associated proteins like ATP synthase components, inclusion of appropriate detergents (DDM, CHAPS) throughout purification is critical to maintain native-like structure and function.
How can researchers verify that recombinant Acidothermus cellulolyticus ATP synthase subunit b retains native structure and function?
Verifying native structure and function of recombinant ATP synthase subunit b requires multiple analytical approaches:
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Structural integrity:
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Functional analysis:
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Molecular interaction studies:
When analyzing results, remember that the ATP synthase b subunit functions as part of a complex, and isolated protein may exhibit different properties than when integrated into the holoenzyme.
What statistical approaches are appropriate for analyzing thermal stability data of ATP synthase components?
When analyzing thermal stability data for ATP synthase components from thermophilic organisms like Acidothermus cellulolyticus, consider these statistical approaches:
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For thermal denaturation curves:
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Non-linear regression analysis to determine melting temperature (Tm)
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Two-state or multi-state unfolding models depending on the complexity of the denaturation profile
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Calculation of thermodynamic parameters (ΔH, ΔS, ΔG) at different temperatures
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For activity versus temperature data:
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Arrhenius plots to determine activation energy
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Modified Eyring equations to calculate entropy and enthalpy of activation
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Non-linear regression to determine temperature optima and thermal inactivation rates
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For comparative studies:
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ANOVA with post-hoc tests for comparing multiple variants
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Correlation analysis between structural features and stability parameters
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Principal component analysis to identify key determinants of thermostability
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Statistical significance should be established with appropriate p-values (<0.05) and data should be presented with standard deviations or standard errors from at least three independent experiments to ensure reproducibility.
How might directed evolution approaches be applied to enhance properties of Acidothermus cellulolyticus ATP synthase subunit b?
Directed evolution offers powerful approaches to engineer enhanced properties in the ATP synthase subunit b:
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Random mutagenesis using error-prone PCR to generate libraries with varying mutation rates
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DNA shuffling between b subunits from different thermophilic organisms to create chimeric proteins with novel properties
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Selection strategies:
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Complementation of b subunit-deficient strains under stress conditions
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Growth-based selection at increasing temperatures
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Activity-based screening in reconstituted systems
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A methodology similar to that used for other thermophilic enzymes could be applied, where random mutagenesis has been used to increase low-temperature activity while maintaining thermostability . This approach could yield variants with broader temperature ranges or enhanced stability at extreme pH or in the presence of denaturants.
What are the potential applications of understanding the structure-function relationship of ATP synthase b subunit in biotechnology?
Understanding the structure-function relationship of the ATP synthase b subunit from thermophilic organisms has several biotechnological applications:
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Designing stabilized energy-transducing membrane proteins for bioenergy applications
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Engineering ATP synthase components for incorporation into synthetic biology systems that require operation at elevated temperatures
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Developing principles for thermostabilizing other membrane proteins of biotechnological interest
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Creating novel nanomotors based on the ATP synthase architecture for nanotechnology applications
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Applying knowledge of peripheral stalk architecture to design stable protein scaffolds for biotechnology
The thermostable nature of Acidothermus cellulolyticus proteins makes them particularly valuable as starting points for protein engineering efforts, as demonstrated by the successful application of other enzymes from this organism in biomass conversion and biofuel production .
