Recombinant Mesorhizobium sp. ATP synthase subunit b 1 (atpF1)

What is ATP synthase subunit b 1 (atpF1) in Mesorhizobium species?

ATP synthase subunit b 1 (atpF1) is a critical component of the F-type ATP synthase complex in Mesorhizobium species. It functions as part of the F0 sector, which is embedded in the membrane and forms the proton channel. The protein is alternatively known as ATP synthase F0 sector subunit b 1, ATPase subunit I 1, or F-ATPase subunit b 1. In Mesorhizobium sp., atpF1 plays an essential role in the energy conversion process, coupling proton translocation across the membrane with ATP synthesis or hydrolysis . This protein contributes to the stator structure that connects the F1 catalytic domain with the F0 membrane sector, allowing the enzyme to function properly in bacterial energy metabolism.

How does atpF1 contribute to ATP synthesis mechanism?

ATP synthase subunit b 1 (atpF1) plays a crucial structural and functional role in the ATP synthesis mechanism. It forms part of the peripheral stalk (or stator) that connects the membrane-embedded F0 sector to the catalytic F1 sector. This connection is vital because it allows the F0 sector to rotate in response to proton movement while keeping the catalytic subunits stationary. The process functions as follows:

• Protons flow through the F0 sector due to the proton gradient across the membrane

• This flow causes rotation of certain parts of the F0 complex

• The rotation energy is transferred to the F1 sector, where it drives conformational changes

• These conformational changes facilitate ATP synthesis from ADP and inorganic phosphate (Pi)

The atpF1 subunit specifically helps maintain the proper orientation and stability of the entire complex during this process, ensuring efficient energy conversion . Recent studies with similar F0F1 ATP synthases have shown that they can interact with polyphosphates, suggesting possible additional roles in bacterial phosphate metabolism beyond ATP synthesis .

How is the recombinant form of atpF1 typically produced?

Recombinant Mesorhizobium sp. ATP synthase subunit b 1 (atpF1) is typically produced using E. coli expression systems. According to product specifications, these recombinant proteins are commonly expressed in E. coli as the host organism . The production process typically involves:

• Cloning the atpF1 gene from Mesorhizobium sp. into an appropriate expression vector

• Transforming the construct into an E. coli expression strain

• Inducing protein expression under optimized conditions

• Purifying the recombinant protein using affinity chromatography

• Confirming identity and purity through SDS-PAGE and other analytical methods

The commercially available recombinant atpF1 protein shows a purity of >85% as determined by SDS-PAGE analysis . When working with this recombinant protein, researchers should be aware that the specific tag type may vary and will be determined during the manufacturing process, potentially affecting experimental design considerations.

What are the optimal storage conditions for recombinant atpF1?

The stability and shelf life of recombinant Mesorhizobium sp. ATP synthase subunit b 1 (atpF1) depend on several factors including storage state, buffer ingredients, storage temperature, and the inherent stability of the protein itself. Based on manufacturer recommendations, the following storage guidelines should be followed:

• For long-term storage, keep the protein at -20°C to -80°C

• The shelf life of the lyophilized form is approximately 12 months at -20°C to -80°C

• The shelf life of the liquid form is approximately 6 months at -20°C to -80°C

• For working aliquots, store at 4°C for up to one week

• Avoid repeated freezing and thawing cycles as this significantly reduces protein activity

For optimal stability, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being typical) before aliquoting for long-term storage. This helps prevent damage from freeze-thaw cycles and maintains protein integrity over time .

How should recombinant atpF1 be reconstituted for experimental use?

Proper reconstitution of recombinant Mesorhizobium sp. ATP synthase subunit b 1 (atpF1) is critical for maintaining its biological activity in experimental applications. The following protocol is recommended:

• Briefly centrifuge the vial containing lyophilized protein prior to opening to bring the contents to the bottom

• Reconstitute the protein in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• For long-term storage of the reconstituted protein, add glycerol to a final concentration of 5-50% (manufacturer's default is 50%)

• Aliquot the glycerol-containing solution into smaller volumes to avoid repeated freeze-thaw cycles

• Store aliquots at -20°C to -80°C for long-term stability

It is important to note that the protein should not undergo repeated freezing and thawing, as this can significantly compromise its activity. For working solutions intended for immediate use, store at 4°C for no longer than one week .

What analytical methods can be used to study atpF1 interactions with other molecules?

Several analytical methods can be employed to study the interactions of recombinant Mesorhizobium sp. ATP synthase subunit b 1 (atpF1) with other molecules:

Molecular Docking Analysis:
Computational docking studies can predict binding interactions between atpF1 and potential ligands. Similar studies with F0F1 ATP synthase have revealed that molecules like polyphosphate (polyP) can bind to the ADP binding site with significant binding affinity (binding scores of -9.3 to -9.6 using AutoDock Vina) . This approach provides valuable insights before conducting wet-lab experiments.

Enzyme Activity Assays:

• ATP hydrolysis assays can measure the impact of potential binding partners on atpF1 function

• pH-sensitive assays can detect proton movement associated with ATP synthase activity

• Polarographic oxygen electrode measurements can assess respiratory changes in subcellular fractions containing ATP synthase

Binding and Structural Studies:

• Electrophoretic mobility shift assays (EMSA) can detect protein-ligand interactions

• DNase I footprinting can identify binding sites if studying nucleic acid interactions

• Site-directed mutagenesis can confirm the importance of specific residues in binding interactions

Advanced Biophysical Techniques:

• Immunocapture of the ATP synthase complex followed by interaction studies

• Surface plasmon resonance (SPR) to measure binding kinetics

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

These methods, alone or in combination, can provide comprehensive insights into how atpF1 interacts with other components of the ATP synthase complex or with regulatory molecules.

How can atpF1 be used to study bacterial energy metabolism?

Recombinant Mesorhizobium sp. ATP synthase subunit b 1 (atpF1) serves as a valuable tool for investigating bacterial energy metabolism through several experimental approaches:

Reconstitution Studies:
Researchers can incorporate purified recombinant atpF1 into liposomes or nanodiscs along with other ATP synthase subunits to create a minimal functional system. This approach allows for controlled examination of how specific mutations or modifications of atpF1 affect proton translocation and ATP synthesis efficiency.

Respiration Analysis:
In systems containing ATP synthase, addition of ADP typically increases respiration rate (state V3) as F0F1 ATP synthase utilizes the proton gradient to synthesize ATP, which then decreases to state V4 when ADP is depleted. Similar experiments with polyphosphate have shown that polyP can also stimulate respiration through interaction with F0F1 ATP synthase, suggesting complex roles in energy metabolism . Recombinant atpF1 can be used in such comparative studies to elucidate its specific contribution to these processes.

Proton Translocation Assessment:
Detailed analysis of atpF1's role in proton movement can be conducted using:

• pH electrodes to detect proton consumption or release

• Fluorescent pH indicators to visualize proton movement in real-time

• Sub-mitochondrial particles or bacterial membrane vesicles with incorporated recombinant atpF1

A typical experimental setup for studying proton translocation involves sub-mitochondrial particles where ATP hydrolysis by F0F1 ATP synthase leads to proton pumping and acidification of the medium. This acidification can be measured using pH electrodes and is sensitive to specific inhibitors like oligomycin .

What is the relationship between atpF1 and polyphosphate metabolism in bacteria?

Recent research has revealed intriguing connections between ATP synthase components like atpF1 and bacterial polyphosphate (polyP) metabolism:

Direct Interaction Evidence:
Molecular docking experiments using F0F1 ATP synthase structures have demonstrated that polyP can bind specifically to the ADP binding site of the F1 region with significant binding affinity (AutoDock Vina binding score -9.3 to -9.6) . This suggests that ATP synthase components, including atpF1, may directly interact with polyP molecules.

Functional Significance:

• F0F1 ATP synthase can both synthesize and hydrolyze polyP in an oligomycin-dependent manner

• This activity occurs at the same catalytic sites used for ATP synthesis/hydrolysis

• The interaction appears specific to the ADP binding region of the enzyme

Experimental Approaches:
Researchers investigating this relationship can employ several methods:

• Acidulation assays with sub-mitochondrial particles to detect proton release during polyP hydrolysis

• Immunocapture techniques to isolate pure F0F1 ATP synthase for direct polyP interaction studies

• Site-directed mutagenesis of key residues in atpF1 and other subunits to assess their role in polyP binding

Significance Table: Binding Energies of Different Molecules to F0F1 ATP Synthase

This relationship suggests that ATP synthase, including its atpF1 component, may play previously unrecognized roles in bacterial phosphate metabolism beyond its classical function in ATP synthesis .

How does atpF1 contribute to the structural stability of ATP synthase?

The ATP synthase subunit b 1 (atpF1) plays a critical role in maintaining the structural integrity and functional stability of the entire ATP synthase complex through several key mechanisms:

Stator Function:
AtpF1 forms part of the peripheral stalk (stator) that connects the membrane-embedded F0 sector with the catalytic F1 sector. This connection is crucial because it prevents the entire F1 sector from rotating when the central rotor turns during ATP synthesis or hydrolysis. Without this stabilizing function, the energy from proton movement could not be effectively converted to the mechanical energy needed for ATP synthesis.

Conformational Flexibility:
The atpF1 subunit exhibits specific structural features that allow it to:

• Maintain a rigid connection between F0 and F1 sectors

• Withstand torque generated during rotational catalysis

• Adapt to different conformational states during the catalytic cycle

Interaction Surface:
The atpF1 subunit provides extensive interaction surfaces for other components of the ATP synthase complex. These interactions are critical for:

• Initial assembly of the complex

• Maintenance of proper subunit orientation during function

• Stability under varying physiological conditions

Studies with F0F1 ATP synthase structures, including docking experiments, have shown that perturbations in these structural roles can significantly impact both the assembly and catalytic efficiency of the entire complex . Researchers investigating atpF1 function can use recombinant protein in reconstitution experiments to assess how specific mutations affect these structural roles.

What are common issues when working with recombinant atpF1 and how can they be resolved?

Researchers working with recombinant Mesorhizobium sp. ATP synthase subunit b 1 (atpF1) may encounter several challenges. The following table outlines common issues and their solutions:

How can researchers validate the structural integrity of recombinant atpF1?

Ensuring the structural integrity of recombinant Mesorhizobium sp. ATP synthase subunit b 1 (atpF1) is crucial for meaningful experimental outcomes. Several complementary approaches can be employed:

Biochemical Assessment:

• SDS-PAGE analysis can confirm the expected molecular weight and initial purity (>85% purity is typical for commercial preparations)

• Native PAGE can help assess the oligomeric state of the protein

• Size exclusion chromatography can reveal aggregation or degradation issues

Biophysical Characterization:

• Circular dichroism (CD) spectroscopy can confirm proper secondary structure content

• Fluorescence spectroscopy can assess tertiary structure integrity

• Differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF) can evaluate thermal stability and folding

Functional Validation:

• Binding assays with known interaction partners (e.g., other ATP synthase subunits)

• Limited proteolysis to assess the presence of properly folded domains resistant to digestion

• Activity assays if the isolated subunit retains measurable function

Structural Analysis:
For more detailed structural validation, researchers can employ:

It's important to validate structural integrity using multiple orthogonal methods rather than relying on a single technique, especially when planning complex interaction studies or functional assays.

What controls should be included in experiments using recombinant atpF1?

Designing appropriate controls is essential for experiments involving recombinant Mesorhizobium sp. ATP synthase subunit b 1 (atpF1). The following controls should be considered for different experimental scenarios:

For Binding/Interaction Studies:

• Negative Control: Buffer-only or irrelevant protein of similar size to rule out non-specific interactions

• Competition Control: Unlabeled atpF1 to compete with labeled atpF1 in binding assays

• Known Binder Control: If available, use a known binding partner as a positive control

• Tag-Only Control: If the recombinant protein contains tags, test the tag alone to ensure it's not responsible for observed interactions

For Functional Assays:

• Inhibitor Control: Use specific ATP synthase inhibitors like oligomycin to confirm specificity of observed activities

• Heat-Inactivated Control: Compare results with heat-denatured atpF1 to confirm that activity requires properly folded protein

• Substrate Specificity Controls: Test related but non-physiological substrates to confirm specificity

For Structural Studies:

• Reference Protein Controls: Include well-characterized proteins of known structure for comparison

• Buffer Matching Controls: Ensure identical buffer conditions between sample and control measurements

• Concentration Series: Test multiple protein concentrations to identify concentration-dependent artifacts

For Reconstitution Experiments:

• Partial Complex Controls: Test systems with specific subunits omitted to determine their contribution

• Orientation Controls: Verify proper orientation of atpF1 when reconstituted into membranes or liposomes

• Activity Benchmarks: Compare activity of reconstituted systems with native membranes or purified complexes

Docking experiments with F0F1 ATP synthase have demonstrated the importance of proper controls. For example, when validating polyP binding to the ADP site, researchers confirmed specificity by showing that docked ADP molecules adopt the same position as those in crystal structures .

What are emerging research areas involving atpF1 in bacterial systems?

Several promising research directions involving Mesorhizobium sp. ATP synthase subunit b 1 (atpF1) are emerging in bacterial systems:

Novel Energy Storage Mechanisms:
Recent findings suggest that ATP synthase components may play roles in polyphosphate metabolism beyond traditional ATP synthesis. Docking experiments have shown that polyP binds to ADP binding sites of F0F1 ATP synthase with high affinity, suggesting a direct role in phosphate storage and energy conversion . Future research could investigate how atpF1 specifically contributes to these alternative energy pathways in Mesorhizobium species.

Cross-talk with Quorum Sensing:
Evidence from Mesorhizobium japonicum indicates complex regulatory networks involving quorum sensing . Investigating potential cross-talk between energy metabolism components like atpF1 and bacterial communication systems could reveal how energy production is integrated with population-level behaviors in soil bacteria and plant symbionts.

Structural Adaptations for Environmental Niches:
Mesorhizobium species occupy specialized ecological niches, often as plant symbionts. Comparative studies of atpF1 structure and function across different Mesorhizobium species could reveal adaptations to different host plants or soil conditions.

Synthetic Biology Applications:
The modular nature of ATP synthase components makes them attractive targets for synthetic biology approaches:

• Engineering optimized energy production systems for biotechnology

• Creating hybrid complexes with novel functionalities

• Developing biosensors based on ATP synthase activity changes

Antimicrobial Resistance Mechanisms:
As ATP synthase is essential for bacterial survival, understanding unique features of atpF1 in Mesorhizobium compared to pathogenic bacteria could inform the development of selective antimicrobials that target specific bacterial groups while sparing beneficial soil bacteria.

How might atpF1 studies contribute to understanding bacterial adaptation to environmental stresses?

Research on Mesorhizobium sp. ATP synthase subunit b 1 (atpF1) offers significant insights into bacterial adaptation mechanisms:

Energy Conservation Under Stress:
During environmental stress, bacteria must carefully manage energy resources. The atpF1 subunit, as part of ATP synthase, plays a crucial role in maintaining energy homeostasis under challenging conditions. Studies of how atpF1 structure and function change under different stress conditions can reveal adaptive mechanisms such as:

• Conformational changes that alter proton translocation efficiency

• Modified interactions with other ATP synthase subunits

• Shifts between ATP synthesis and ATP hydrolysis modes depending on environmental conditions

Integration with Stress Response Pathways:
AtpF1 function likely integrates with broader stress response networks:

• Transcriptional regulation of atpF1 expression under different stresses

• Post-translational modifications that alter function in response to environmental signals

• Protein-protein interactions that change under stress conditions

Role in Polyphosphate-Mediated Stress Resistance:
The discovered connection between F0F1 ATP synthase and polyphosphate metabolism suggests that atpF1 may participate in stress adaptation through polyP-related mechanisms:

• Accumulation of polyP as an energy reserve during nutrient limitation

• Use of polyP for protein phosphorylation during stress signaling

• Chelation of toxic metals by polyP as a protective mechanism

Methodological Approaches for Stress Studies:
Researchers investigating atpF1 in stress responses might employ:

• Comparative proteomics to identify stress-induced modifications

• In vitro reconstitution under varying pH, temperature, or ionic conditions

• Binding studies with stress-induced metabolites

• Gene expression analysis under different environmental challenges

Understanding these mechanisms could provide insights into how beneficial soil bacteria like Mesorhizobium survive environmental fluctuations, with potential applications for improving plant-microbe interactions in changing climate conditions.