Recombinant Aliivibrio salmonicida ATP synthase subunit b (atpF)

What is Aliivibrio salmonicida ATP synthase subunit b (atpF) and what is its biological significance?

Aliivibrio salmonicida ATP synthase subunit b (atpF) is a critical component of the F0 sector of the F1F0 ATP synthase complex in this psychrophilic fish pathogen. The protein functions as part of the peripheral stalk (stator) that connects the membrane-embedded F0 sector with the catalytic F1 sector of the ATP synthase. This connection is essential for the mechanical coupling between proton translocation across the membrane and ATP synthesis. As A. salmonicida is the causative agent of cold water vibriosis affecting farmed fish species, its energy metabolism components, including ATP synthase, represent important adaptive features for survival in cold environments .

The biological significance of this protein extends beyond its structural role in ATP synthesis. As a component from a psychrophilic organism, it likely contains cold-adaptive features that allow it to maintain flexibility and function at lower temperatures compared to mesophilic homologs. These adaptations make it a valuable model for studying protein evolution in response to environmental pressures.

How does Aliivibrio salmonicida ATP synthase subunit b differ from homologous proteins in mesophilic organisms?

While the search results don't provide direct comparative data between A. salmonicida ATP synthase subunit b and mesophilic homologs, general principles of cold adaptation in proteins suggest several likely differences:

• Amino acid composition: Psychrophilic proteins typically contain fewer proline and arginine residues (which increase rigidity) and more glycine residues (which increase flexibility) compared to mesophilic homologs.

• Surface charge distribution: Cold-adapted proteins often display an increased proportion of negatively charged residues on their surface, which can reduce the strength of hydrophobic interactions and increase flexibility.

• Reduced structural stability: The protein likely exhibits reduced hydrophobic core packing and fewer stabilizing interactions such as salt bridges and hydrogen bonds, allowing for greater flexibility at lower temperatures.

• Increased activity at low temperatures: Structural modifications likely result in lower activation energy for conformational changes necessary for function, enabling the protein to maintain activity in cold environments where mesophilic homologs would become too rigid.

These adaptations would be particularly important for ATP synthase components, as they must maintain both structural integrity and conformational flexibility to support the rotary mechanism of ATP synthesis in cold environments.

What expression systems are most effective for producing recombinant Aliivibrio salmonicida ATP synthase subunit b?

Based on research with similar proteins, Escherichia coli expression systems have proven effective for producing recombinant proteins from Aliivibrio salmonicida. Several expression vectors warrant consideration for optimal results:

• pMAL-c2x vector system: This system allows expression of the target protein as a fusion with maltose-binding protein (MBP), which has been successfully used for other A. salmonicida proteins to enhance solubility and reduce toxicity to host cells . The MBP tag also provides a convenient purification handle via amylose affinity chromatography.

• pET expression system: The pET-32a(+) vector, which includes thioredoxin fusion capabilities, has been used successfully for ATP synthase subunit expression . The T7 promoter-based system provides high-level, inducible expression when the host strain contains a chromosomal copy of T7 RNA polymerase.

• pFLAG-MAC vector: This system allows expression with a FLAG epitope tag, facilitating detection and purification via anti-FLAG affinity chromatography .

For optimal expression of membrane-associated proteins like ATP synthase subunit b, expression parameters including temperature (typically lowered to 16-20°C for psychrophilic proteins), IPTG concentration (0.1-0.5 mM), and induction duration (overnight or longer at reduced temperatures) must be carefully optimized to balance expression level with proper folding.

What strategies can overcome common challenges in expressing recombinant Aliivibrio salmonicida ATP synthase subunit b?

Expression of recombinant A. salmonicida ATP synthase subunit b presents several challenges that can be addressed with the following strategies:

Table 1. Challenges and Solutions for Expression of Recombinant A. salmonicida ATP synthase subunit b

Specifically, the fusion to large solubility tags like MBP has proven effective for other A. salmonicida proteins that exhibited toxicity to E. coli cells . Additionally, co-expression with the pOFXT7KJE3 plasmid encoding chaperone proteins DnaK, DnaJ, and GrpE has been shown to substantially increase yields of difficult-to-express proteins .

What is the optimal purification protocol for obtaining high-purity recombinant Aliivibrio salmonicida ATP synthase subunit b?

A multi-step purification protocol incorporating complementary chromatographic techniques would yield the highest purity recombinant A. salmonicida ATP synthase subunit b:

• Initial affinity chromatography: Based on the fusion tag used for expression. For MBP-tagged protein, amylose resin affinity chromatography would be appropriate, with elution using maltose buffer. For His-tagged protein, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resin would be suitable.

• Nucleic acid removal: Treatment with a nuclease inhibitable by reducing agents is essential to remove bacterial DNA without introducing contaminating nucleases that might interfere with downstream applications . Commercially available nucleases like Benzonase can be used, followed by addition of reducing agents (e.g., DTT or β-mercaptoethanol) to inhibit the nuclease activity.

• Tag removal: Cleavage of the fusion tag using an appropriate protease (e.g., TEV protease for a TEV cleavage site, Factor Xa for MBP fusion proteins, or thrombin for GST fusion proteins), followed by a second affinity step to remove the cleaved tag.

• Ion exchange chromatography: Based on the theoretical isoelectric point of the protein, either cation or anion exchange chromatography would serve as an effective secondary purification step.

• Size exclusion chromatography: As a final polishing step to remove any remaining contaminants, aggregates, or degradation products, and to exchange the protein into the final storage buffer.

Throughout the purification process, it is essential to maintain conditions that stabilize the protein, potentially including detergents or lipids to stabilize the transmembrane domain, and consider the temperature sensitivity of this psychrophilic protein.

How can the ATP synthase activity of recombinant Aliivibrio salmonicida ATP synthase subunit b be measured?

Measuring the ATP synthase activity requires reconstitution of the entire ATP synthase complex or functional studies within the context of the assembled complex. Although the b subunit alone doesn't have enzymatic activity, its contribution to ATP synthase function can be assessed through several approaches:

• Reconstitution studies: The recombinant A. salmonicida ATP synthase subunit b can be reconstituted with other subunits to form the complete ATP synthase complex. Similar to studies with the F1F0 ATP synthase β subunit , this would involve:

  • Assembly of the complex from individual purified subunits

  • Incorporation into liposomes to create a proton gradient

  • Measurement of ATP synthesis or hydrolysis rates using luciferase-based ATP detection or phosphate release assays

• ATPase activity assays: Once reconstituted, the ATPase activity of the complex can be measured using established methods to quantify either ATP hydrolysis (reverse direction) or ATP synthesis (forward direction) . The impact of the b subunit can be assessed by comparing complexes with wild-type versus mutant versions of the subunit.

• Proton pumping assays: Using pH-sensitive fluorescent dyes like ACMA (9-amino-6-chloro-2-methoxyacridine) to measure proton translocation across liposomal membranes containing reconstituted ATP synthase complexes.

• Temperature-dependent activity profiles: Given A. salmonicida's psychrophilic nature, measuring activity across a temperature range (0-37°C) would provide valuable insights into cold adaptation of the ATP synthase complex.

What techniques are most effective for studying protein-protein interactions involving Aliivibrio salmonicida ATP synthase subunit b?

Several complementary techniques can be employed to study protein-protein interactions involving A. salmonicida ATP synthase subunit b:

• Co-immunoprecipitation (Co-IP): Using antibodies against the b subunit or its fusion tag to precipitate the protein along with its interaction partners from cell lysates or reconstituted systems.

• Pull-down assays: Similar to Co-IP but using the affinity tag (MBP, GST, or His) for capture of the recombinant protein and its interacting partners.

• Surface plasmon resonance (SPR): For quantitative measurement of binding kinetics between the b subunit and other ATP synthase components, providing association and dissociation rate constants as well as binding affinity.

• Isothermal titration calorimetry (ITC): To measure the thermodynamic parameters of protein-protein interactions, particularly valuable for studies of cold adaptation.

• Chemical cross-linking coupled with mass spectrometry: To identify interaction interfaces between the b subunit and other components of the ATP synthase complex. This approach has been particularly useful for studying membrane protein complexes.

• Fluorescence resonance energy transfer (FRET): By labeling the b subunit and potential interaction partners with fluorescent dyes, FRET can detect interactions in real-time and in a native-like environment.

• Yeast two-hybrid or bacterial two-hybrid systems: Although these may be challenging for membrane proteins, modified versions designed for membrane proteins could potentially be used to screen for interaction partners.

These methods can be particularly valuable for comparing interactions at different temperatures to understand how the psychrophilic nature of A. salmonicida influences the assembly and stability of the ATP synthase complex.

How does temperature affect the structure and function of Aliivibrio salmonicida ATP synthase subunit b?

As a component from a psychrophilic organism, A. salmonicida ATP synthase subunit b likely exhibits specific structural and functional adaptations to low temperatures. While the search results don't provide direct experimental data on temperature effects, the following approaches would be valuable for investigating these adaptations:

• Circular dichroism (CD) spectroscopy: To monitor secondary structure content and stability at different temperatures, providing insights into cold-adaptive features such as increased flexibility or reduced thermal stability.

• Differential scanning calorimetry (DSC): To measure the thermodynamic parameters of protein unfolding at different temperatures, quantifying stability differences between A. salmonicida ATP synthase subunit b and mesophilic homologs.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): To assess protein dynamics and flexibility at different temperatures, potentially revealing regions with enhanced flexibility that contribute to cold adaptation.

• Temperature-dependent activity assays: Measuring ATP synthase activity of reconstituted complexes across a temperature range to establish the optimal temperature for activity and the temperature range within which the complex remains functional.

• Molecular dynamics simulations: Computational approaches to model the dynamics of the protein at different temperatures, comparing with mesophilic homologs to identify structural features contributing to cold adaptation.

These studies would likely reveal that A. salmonicida ATP synthase subunit b maintains structural integrity and functional capability at temperatures where mesophilic homologs become rigid and inactive, reflecting evolutionary adaptations to the cold marine environment where this fish pathogen resides.

What structural biology techniques are most suitable for studying recombinant Aliivibrio salmonicida ATP synthase subunit b?

Multiple structural biology techniques can provide complementary information about A. salmonicida ATP synthase subunit b structure:

These techniques would ideally be combined with computational approaches such as molecular modeling and molecular dynamics simulations to develop a comprehensive understanding of the protein's structure and dynamics, particularly in relation to cold adaptation.

How can site-directed mutagenesis of recombinant Aliivibrio salmonicida ATP synthase subunit b inform structure-function relationships?

Site-directed mutagenesis represents a powerful approach for investigating structure-function relationships in A. salmonicida ATP synthase subunit b, similar to studies conducted with the β subunit of ATP synthase . The following systematic mutagenesis strategies would be particularly informative:

• Alanine-scanning mutagenesis: Systematically replacing key residues with alanine to identify amino acids essential for:

  • Protein stability and folding

  • Interactions with other ATP synthase components

  • Cold adaptation features

• Charge-swap mutations: Modifying charged residues to investigate the role of electrostatic interactions in protein stability and complex assembly, particularly relevant for cold adaptation where electrostatic interactions are often modified.

• Phosphomimetic mutations: Creating phosphomimetic (e.g., serine/threonine to glutamate) and non-phosphorylatable (e.g., serine/threonine to alanine) variants to investigate potential regulatory phosphorylation sites, similar to studies with the β subunit .

• Chimeric proteins: Creating chimeras between A. salmonicida ATP synthase subunit b and homologs from mesophilic organisms to identify regions responsible for cold adaptation.

• Flexibility-modifying mutations: Targeting regions predicted to have enhanced flexibility in this psychrophilic protein to determine the impact on function at different temperatures.

The functional impact of these mutations could be assessed through:

• Complex formation analysis using native PAGE or size exclusion chromatography

• ATPase activity assays of reconstituted complexes

• Structural stability assessments using thermal denaturation studies

• Protein-protein interaction analyses using techniques described in section 3.2

This approach has proven informative with the ATP synthase β subunit, where phosphomimetic mutations at specific residues (e.g., T262E) abolished activity while non-phosphorylatable variants maintained wild-type activity levels .

What computational approaches can predict cold-adaptive features of Aliivibrio salmonicida ATP synthase subunit b?

Several computational approaches can predict and analyze cold-adaptive features of A. salmonicida ATP synthase subunit b:

• Comparative sequence analysis: Alignment of A. salmonicida ATP synthase subunit b with homologs from organisms adapted to different temperature ranges to identify signature sequence patterns associated with cold adaptation, such as:

  • Reduced proline content

  • Increased glycine content

  • Reduced arginine/increased lysine ratio

  • Higher surface-exposed acidic residue content

• Homology modeling: Generation of three-dimensional structural models based on crystal structures of homologous proteins, followed by analysis of:

  • Surface charge distribution

  • Hydrophobic core packing

  • Distribution of stabilizing interactions (hydrogen bonds, salt bridges)

  • Prediction of regions with enhanced flexibility

• Molecular dynamics (MD) simulations: Simulation of protein behavior at different temperatures to analyze:

  • Temperature-dependent conformational flexibility

  • Stability of secondary structure elements

  • Water interactions and solvation patterns

  • Behavior of the protein in membrane environments

• Energy calculation methods: Computational assessment of the thermodynamic stability of the protein structure through:

  • Free energy calculations

  • Electrostatic potential mapping

  • Hydrogen bond network analysis

  • Prediction of weak points in the structure at increased temperatures

• Machine learning approaches: Training models on known cold-adapted proteins to identify features associated with psychrophilic adaptation and applying these models to predict cold-adaptive features in A. salmonicida ATP synthase subunit b.

These computational approaches, when combined with experimental validation, can provide valuable insights into the molecular basis of cold adaptation in A. salmonicida ATP synthase subunit b and guide rational design of experiments to further investigate these features.

How can recombinant Aliivibrio salmonicida ATP synthase subunit b be used to study ATP synthase assembly in psychrophilic organisms?

Recombinant A. salmonicida ATP synthase subunit b provides a valuable tool for investigating ATP synthase assembly in psychrophilic organisms through several experimental approaches:

• In vitro reconstitution studies: Using purified recombinant components to reconstruct the ATP synthase complex under controlled conditions, similar to approaches used with ATP synthase subunit c . This would allow investigation of:

  • The order of assembly steps

  • Temperature dependence of assembly

  • Minimum components required for stable complex formation

  • Role of lipids in the assembly process

• Fluorescently labeled subunit tracking: Tagging the recombinant subunit b with fluorescent proteins or dyes to visualize its incorporation into the ATP synthase complex in real-time, potentially using techniques like fluorescence recovery after photobleaching (FRAP) to study the dynamics of assembly.

• Heterologous expression systems: Expressing A. salmonicida ATP synthase subunit b in mesophilic hosts at different temperatures to investigate compatibility with host ATP synthase components and temperature constraints on assembly.

• Assembly intermediates capture: Using crosslinking approaches to capture and characterize assembly intermediates, providing insights into the assembly pathway and potential differences from mesophilic systems.

• Chaperone requirements: Investigating the potential role of chaperone proteins in facilitating the assembly of A. salmonicida ATP synthase, particularly at low temperatures. Co-expression with chaperones like those encoded by the pOFXT7KJE3 plasmid (DnaK, DnaJ, and GrpE) could be especially informative .

These studies would contribute to our understanding of how essential multiprotein complexes assemble in cold environments and how this process has been adapted during evolution of psychrophilic organisms.

What insights can Aliivibrio salmonicida ATP synthase subunit b provide about the evolution of psychrophilic adaptations?

Evolutionary analysis of A. salmonicida ATP synthase subunit b can provide significant insights into psychrophilic adaptation mechanisms:

• Phylogenetic analysis: Comparing ATP synthase subunit b sequences across the Vibrionaceae family and related bacteria adapted to different temperature ranges would reveal evolutionary patterns associated with temperature adaptation. This would allow identification of:

  • Conserved residues essential for function across all temperature ranges

  • Residues specifically modified in cold-adapted lineages

  • Convergent evolutionary changes in distantly related psychrophilic organisms

• Molecular clock analyses: Estimating the timing of adaptive changes in relation to environmental cooling events or habitat transitions could reveal how rapidly ATP synthase components evolve in response to temperature changes.

• Selection pressure analysis: Calculating the ratio of non-synonymous to synonymous substitutions (dN/dS) would identify residues under positive selection pressure during adaptation to cold environments.

• Ancestral sequence reconstruction: Computational prediction of ancestral protein sequences followed by experimental characterization of resurrected proteins would provide direct evidence of the functional impacts of evolutionary changes.

• Horizontal gene transfer assessment: Analysis of anomalous sequence patterns or phylogenetic incongruencies could reveal potential instances of horizontal gene transfer that might have contributed to rapid adaptation to cold environments.

These evolutionary insights would extend beyond A. salmonicida ATP synthase subunit b to enhance our understanding of general principles governing protein adaptation to extreme environments and potentially inform protein engineering efforts aimed at modifying temperature optima of industrial enzymes.

What potential applications exist for using recombinant Aliivibrio salmonicida ATP synthase subunit b in biotechnology and medicine?

Recombinant A. salmonicida ATP synthase subunit b presents several potential applications in biotechnology and medicine:

• Vaccine development: As A. salmonicida causes cold water vibriosis in farmed fish species, recombinant ATP synthase components could potentially serve as vaccine antigens. The search results indicate that while vibriosis is "fully controlled by vaccination," the molecular mechanisms behind the successful vaccine remain largely unknown . ATP synthase components, being highly conserved and essential for bacterial survival, represent potential vaccine targets.

• Antibiotic development: The essential nature of ATP synthase for bacterial survival makes it a potential target for new antibiotics. Structural and functional studies of A. salmonicida ATP synthase subunit b could inform the development of specific inhibitors targeting the ATP synthase of pathogenic Vibrionaceae.

• Protein engineering platforms: The cold-adaptive features of A. salmonicida ATP synthase subunit b could serve as a template for engineering cold-activity into industrial enzymes. Understanding these adaptations at the molecular level could inform rational design strategies for creating enzymes that function efficiently at low temperatures.

• Biosensors for low-temperature environments: The temperature-sensitive properties of psychrophilic proteins could be exploited to develop biosensors for monitoring cold environments or cold-chain logistics in food and pharmaceutical industries.

• Structural biology tools: The unique properties of psychrophilic proteins, such as enhanced flexibility and reduced stability, could make them valuable tools for structural biology studies, potentially facilitating crystallization or providing model systems for studying protein dynamics.

These applications build upon the fundamental research value of A. salmonicida ATP synthase subunit b as a model system for understanding cold adaptation mechanisms in essential cellular machinery.