Recombinant Photobacterium profundum Asparagine--tRNA ligase (asnS)

What is Photobacterium profundum and why is it significant for studying asnS function?

Photobacterium profundum SS9 is a Gram-negative bacterium originally collected from the Sulu Sea with a genome consisting of two chromosomes and an 80 kb plasmid. It has become a model organism for studying piezophily (pressure adaptation) because it grows optimally at 28 MPa and 15°C but maintains the ability to grow at atmospheric pressure. This unique characteristic allows for easy genetic manipulation and culture while studying pressure-adapted proteins .

The significance of studying asnS (asparagine-tRNA ligase) in P. profundum lies in understanding how this essential enzyme functions under extreme pressure conditions, potentially revealing novel adaptations in the translation machinery that enable protein synthesis in the deep sea environment.

How does the asnS gene differ between P. profundum and non-pressure adapted organisms?

While the search results don't provide direct comparative data for asnS genes, research on other pressure-adapted genes in P. profundum reveals important principles. P. profundum proteins often contain structural modifications that maintain functionality under high pressure. For example, when P. profundum genes PBPRA3229 (DiaA homolog) and PBPRA1039 (SeqA homolog) were expressed in E. coli, they successfully complemented the corresponding E. coli mutations, demonstrating functional conservation despite adaptation to different pressure environments .

Similar comparative studies would be valuable for understanding asnS adaptations, particularly examining sequence variations in the catalytic and tRNA binding domains that might contribute to pressure resistance while maintaining enzymatic function.

What expression systems are recommended for producing recombinant P. profundum asnS?

Based on methodologies used for similar enzymes, heterologous expression in E. coli systems using vectors with strong, inducible promoters like pET or pBAD series would be appropriate for initial production of recombinant P. profundum asnS. For specific applications requiring native-like conditions, expression in P. profundum itself might be preferable.

The conjugation method described for transferring plasmids from E. coli to P. profundum could also be utilized, where E. coli donors and P. profundum acceptor strains are mixed in a 1:1 ratio, plated on marine agar, and incubated at 20°C for approximately 40 hours .

How should I design experiments to compare asnS activity under different pressure conditions?

When designing experiments to evaluate asnS activity under varying pressure conditions, consider implementing a Completely Randomized Design (CRD) where the independent variable is hydrostatic pressure and the dependent variable is asnS enzymatic activity. The mathematical model would be:

$Y_{ij} = \mu + \tau_i + \epsilon_{ij}$

Where:

For enzymatic assays, adapt methods from similar studies, such as the luminescence-based AMP assay used for asparagine synthetase, which provides a reproducible protocol for measuring activity . To maintain experimental validity, include appropriate controls at each pressure point and perform reactions in triplicate.

Pressure conditions should include at minimum:

• Atmospheric pressure (0.1 MPa)

• Optimal growth pressure for P. profundum (28 MPa)

• Intermediate and higher pressure points (e.g., 10 MPa and 40 MPa)

What purification methods are most effective for recombinant P. profundum asnS?

Based on methodologies for similar enzymes, an effective purification protocol for recombinant P. profundum asnS would likely combine affinity chromatography with subsequent polishing steps:

• Express the protein with an affinity tag (His6 or FLAG) to facilitate initial capture

• For FLAG-tagged proteins, use an immunoprecipitation-based purification scheme similar to that described for asparagine synthetase

• For His-tagged constructs, use immobilized metal affinity chromatography (IMAC)

• Follow with size exclusion chromatography to ensure homogeneity

• Confirm purity using SDS-PAGE and Western blotting

When working with pressure-adapted enzymes, it's crucial to maintain appropriate buffer conditions that preserve the native structure. Consider including stabilizing agents like glycerol (10-15%) and reducing agents to prevent oxidation of cysteine residues.

How can I measure the kinetic parameters of asnS under high pressure conditions?

Measuring kinetic parameters under high pressure requires specialized equipment and methodologies:

• Use a high-pressure reaction vessel capable of maintaining stable pressure while allowing sampling or real-time measurement

• Adapt established enzymatic assays to pressure-compatible formats

• For asnS activity, monitor either AMP production (product) or ATP consumption (substrate)

A luminescence-based AMP detection assay, similar to that described for asparagine synthetase, could be adapted for high-pressure studies . The reaction can be quenched at specific time points by rapid decompression and transfer to assay reagents.

For kinetic parameters, measure initial reaction rates across a range of substrate concentrations at each pressure point, then use Michaelis-Menten kinetics to determine Km and Vmax values:

$v = \frac{V_{max}[S]}{K_m + [S]}$

Compare these parameters across pressure conditions to understand how pressure affects substrate binding affinity and catalytic efficiency.

How do mutations in P. profundum asnS affect its function under high pressure?

To investigate the effects of mutations on asnS function under high pressure, consider the structure-function approach used for asparagine synthetase. This would involve:

• Identifying conserved and variant residues through sequence alignment with non-piezophilic homologs

• Creating site-directed mutants focusing on:

  • Catalytic site residues

  • tRNA binding interface

  • Pressure-specific structural elements

• Expressing and purifying wild-type and mutant proteins

• Comparing enzymatic activities at various pressures

Based on studies with human asparagine synthetase variants, where mutations led to 36-96% reductions in activity , similar comparative analysis for P. profundum asnS would be informative. Use protein modeling software (such as UCSF Chimera) to visualize mutation locations relative to functional domains .

A potential data presentation format could be:

How does P. profundum asnS interact with tRNA molecules under high pressure?

Investigating asnS-tRNA interactions under high pressure would require specialized techniques that can detect biomolecular interactions under pressure. Consider these methodological approaches:

• Fluorescence-based binding assays using labeled tRNA

• High-pressure nuclear magnetic resonance (NMR) spectroscopy

• Molecular dynamics simulations parameterized for high-pressure conditions

• Cross-linking experiments performed under pressure followed by mass spectrometry analysis

Building on methodologies from RNA-protein interaction studies, such as those for tRNA(Asp)/aspartyl-tRNA synthetase complex , similar approaches could be adapted for P. profundum asnS. Photoactivatable probes like aryldiazonium salts could be particularly valuable as they enable RNA mapping and footprinting of RNA/protein interactions .

Analysis should focus on identifying pressure-dependent changes in:

• Binding affinity (Kd)

• Contact residues/nucleotides

• Conformational changes in enzyme or tRNA

• Catalytic efficiency of aminoacylation

What proteomics approaches can identify asnS interaction networks in P. profundum?

For investigating the interaction network of asnS in P. profundum, researchers should consider:

• Label-free quantitative proteomics: Similar to the MS-based label-free quantitative proteomics approach used for P. profundum grown under different pressure conditions . This method would allow comparison of protein expression levels between wild-type and asnS-mutant strains.

• Proximity-dependent labeling: Using BioID or APEX2 fused to asnS to identify proximal proteins in vivo under different pressure conditions.

• Affinity purification-mass spectrometry (AP-MS): Using tagged asnS as bait to pull down interaction partners, followed by mass spectrometry identification.

• Crosslinking mass spectrometry (XL-MS): To capture transient interactions under native pressure conditions.

The shotgun proteomic analysis methods described for P. profundum provide a foundation for these approaches. When analyzing results, focus on pressure-dependent changes in the interaction network and connections to translation machinery components.

How does P. profundum asnS compare to homologs from other deep-sea bacteria?

While direct comparative data for asnS across deep-sea bacteria is not provided in the search results, the approach used for comparing other genes can be applied. The suppression subtractive hybridization (SSH) technique described for Photobacterium damselae could be adapted to identify unique regions in asnS genes across piezophilic bacteria.

Key comparative analyses should include:

• Sequence alignment of asnS genes from various piezophilic and non-piezophilic bacteria

• Phylogenetic analysis to determine evolutionary relationships

• Structural modeling to identify pressure-adaptive features

• Functional complementation studies similar to those performed with DiaA and SeqA homologs

Expected findings might include conserved catalytic domains across all homologs but differences in flexible regions or surface residues that contribute to pressure adaptation. These comparative studies could reveal convergent or divergent evolutionary strategies for maintaining aminoacyl-tRNA synthetase function under pressure.

What role does asnS play in P. profundum's adaptation to high-pressure environments?

Understanding asnS's role in pressure adaptation requires connecting its function to broader metabolic and cellular processes in P. profundum. Proteomic analyses have shown that proteins involved in key metabolic pathways exhibit differential expression under varying pressure conditions .

Specifically, proteins involved in glycolysis/gluconeogenesis were up-regulated at high pressure, while several oxidative phosphorylation proteins were up-regulated at atmospheric pressure . The role of asnS should be examined in this context, particularly regarding:

• Potential co-regulation with other pressure-responsive genes

• Contributions to maintaining translation fidelity under pressure

• Possible moonlighting functions beyond canonical aminoacylation

Experimental approaches could include:

• Creating conditional asnS mutants to observe growth phenotypes at different pressures

• Transcriptomic analysis to identify co-regulated genes

• Metabolomic profiling to detect changes in asparagine and related metabolites

Can recombinant P. profundum asnS be used for in vitro translation systems adapted for high pressure?

Developing high-pressure in vitro translation systems using components from piezophilic organisms represents an advanced research direction. Recombinant P. profundum asnS could be a key component in such systems.

Methodological considerations include:

• Reconstituting a minimal translation system with purified components from P. profundum or hybrid systems combining piezophilic and mesophilic components

• Designing pressure-resistant reaction vessels with capabilities for reagent addition and product monitoring

• Optimizing buffer conditions to maintain component stability under pressure

• Comparing translation efficiency and accuracy across pressure ranges

Expected challenges include maintaining the activity of all components (ribosomes, tRNAs, translation factors) under pressure and developing real-time assays compatible with high-pressure conditions.

Potential applications extend beyond fundamental research to biotechnological uses, such as the production of pressure-stable proteins or enzymes through directed evolution in a pressure-adapted translation system.

What are common challenges in expressing and purifying functional P. profundum asnS?

Researchers working with recombinant P. profundum asnS may encounter several challenges:

• Low expression yields: P. profundum proteins may contain rare codons or form inclusion bodies when expressed in E. coli. Consider:

  • Codon optimization for the expression host

  • Co-expression with chaperones

  • Using cold-adapted expression strains or lower induction temperatures

• Loss of activity during purification: Pressure-adapted enzymes may be sensitive to standard purification conditions. Mitigation strategies include:

  • Including stabilizing agents in buffers

  • Maintaining shorter purification workflows

  • Considering detergent addition if membrane association is suspected

• Verification of proper folding: Methods similar to those used for human asparagine synthetase variants can be employed to verify proper protein folding, including:

  • Circular dichroism spectroscopy

  • Limited proteolysis

  • Thermal shift assays

• Difficulty with activity assays: If direct aminoacylation assays prove challenging, consider surrogate assays like ATP consumption or AMP production as demonstrated for asparagine synthetase .

How should experimental conditions be adjusted when working with pressure-adapted enzymes?

When working with pressure-adapted enzymes like P. profundum asnS, consider these adjustments:

• Temperature considerations: Maintain lower temperatures (10-15°C) during purification and assays, as P. profundum grows optimally at 15°C .

• Buffer optimization:

  • Include osmolytes that mimic deep-sea conditions

  • Adjust ionic strength to compensate for pressure effects on ionization

  • Consider the volumetric effects of pressure on buffer pH

• Storage conditions:

  • Test stability at different pressures

  • Determine if flash-freezing affects activity more than for mesophilic homologs

  • Consider lyophilization as an alternative preservation method

• Substrate considerations: Ensure substrates (ATP, asparagine, tRNA) are stable under experimental conditions by including appropriate controls.

• Equipment adaptation: Standard laboratory equipment may need modification for pressure studies, similar to the adaptations required for proteomic analysis of P. profundum under different pressure conditions .

What emerging technologies could advance our understanding of P. profundum asnS function?

Several cutting-edge technologies could significantly advance research on P. profundum asnS:

• Cryo-electron microscopy (cryo-EM) under pressure: Capturing the structure of asnS in complex with tRNAAsn under native pressure conditions could reveal pressure-specific conformational states.

• Single-molecule approaches: Techniques like Förster resonance energy transfer (FRET) adapted for high-pressure conditions could reveal dynamic aspects of enzyme function.

• High-throughput mutagenesis and selection: Developing pressure-based selection systems combined with deep mutational scanning could identify critical residues for pressure adaptation.

• Synthetic biology approaches: Engineering chimeric asnS enzymes combining domains from piezophilic and non-piezophilic organisms could delineate pressure-adaptation determinants.

• Systems biology integration: Combining proteomics, transcriptomics, and metabolomics data, similar to the proteomic approach used for P. profundum , to place asnS function in the broader context of cellular pressure response.

How might findings from P. profundum asnS research be applied to other pressure-adapted systems?

Research on P. profundum asnS has broad implications for understanding biological adaptation to extreme environments:

• Biotechnological applications: Insights into pressure-stable enzymes could inform the design of biocatalysts for industrial processes under high pressure, similar to how DiaA and SeqA homologs from P. profundum were functionally characterized .

• Astrobiology: Understanding how translation machinery adapts to pressure could inform search parameters for life in high-pressure extraterrestrial environments like subsurface oceans on icy moons.

• Evolutionary biology: Comparative studies of aminoacyl-tRNA synthetases across pressure gradients could reveal convergent evolutionary strategies for maintaining protein synthesis under extreme conditions.

• Structural biology principles: Identifying structural features that confer pressure resistance could inform general principles of protein stability under extreme conditions.

• Origin of life theories: Insights into pressure-adapted translation systems might inform hypotheses about the emergence of protein synthesis in deep-sea hydrothermal vent environments.