Recombinant Photobacterium profundum Alanine--tRNA ligase (alaS), partial

What is Photobacterium profundum alanyl-tRNA synthetase and what is its function?

Photobacterium profundum alanyl-tRNA synthetase (AlaRS), encoded by the alaS gene, belongs to the aminoacyl-tRNA synthetase family of enzymes critical for protein translation. This enzyme catalyzes a two-step reaction: first activating alanine through ATP to form an aminoacyl-adenylate intermediate, and then transferring the activated alanine to its cognate tRNA^Ala. Similar to other bacterial AlaRS enzymes, P. profundum AlaRS likely contains distinct domains for aminoacylation and editing functions, comparable to those identified in E. coli AlaRS . The enzyme plays an essential role in maintaining translational fidelity by ensuring the correct attachment of alanine to tRNA^Ala and preventing misaminoacylation with non-cognate amino acids such as glycine and serine.

As P. profundum is a piezophilic bacterium that grows optimally at 28 MPa and 15°C, its AlaRS may possess structural and functional adaptations that enable efficient protein synthesis under high-pressure conditions . These adaptations would be critical to maintain translational accuracy in the deep-sea environment where this organism naturally thrives.

How does P. profundum AlaRS differ from other bacterial AlaRS enzymes?

P. profundum AlaRS likely possesses unique structural adaptations related to its piezophilic lifestyle. While the core catalytic and editing mechanisms are presumably conserved across bacterial species, P. profundum AlaRS may contain pressure-adaptive features such as:

• Altered amino acid composition with increased flexibility in certain regions to maintain activity under high pressure

• Specific substitutions at key positions to stabilize the active site under pressure conditions

• Potentially modified interactions with tRNA^Ala that remain stable at high pressure

E. coli AlaRS contains several cysteine residues, including a critical C666 in the editing domain . P. profundum AlaRS may possess modifications to these residues that affect its response to varying pressure conditions. Additionally, P. profundum's genome has evolved for optimal growth at high pressure, so its AlaRS may contain pressure-adaptive features not found in non-piezophilic bacteria .

What are the recommended methods for cloning and expressing recombinant P. profundum AlaRS?

For successful cloning and expression of recombinant P. profundum AlaRS, the following methodological approach is recommended:

Cloning Strategy:

• Amplify the P. profundum alaS gene using high-fidelity PCR with primers containing appropriate restriction sites

• Clone the amplified gene into an expression vector such as pET21b with a C-terminal His-tag for purification purposes

• Verify the construct through sequencing to confirm the correct insertion and absence of mutations

Expression Protocol:

• Transform the verified construct into an E. coli expression strain such as BL21(DE3)

• Culture transformed cells in LB media until reaching mid-log phase (OD600 ≈ 0.6-0.8)

• Induce protein expression with 1 mM IPTG at 37°C for 4 hours or at lower temperatures (16-18°C) overnight for potentially improved solubility

• Harvest cells by centrifugation and proceed with protein purification

This protocol is adapted from methods used for E. coli AlaRS expression , with modifications to account for potential challenges in expressing a protein from a piezophilic organism.

What is the optimal purification protocol for recombinant P. profundum AlaRS?

The optimal purification protocol for recombinant P. profundum AlaRS involves multiple steps designed to obtain high-purity, active enzyme:

Purification Protocol:

• Resuspend harvested cells in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and protease inhibitors

• Lyse cells by sonication or high-pressure homogenization

• Clarify the lysate by centrifugation at 16,000 × g for 30 minutes at 4°C

• Purify the His-tagged protein using metal affinity chromatography (TALON or Ni-NTA resin)

• Elute the protein with 250 mM imidazole

• Concentrate the eluted fractions and perform buffer exchange by dialysis into a storage buffer containing 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 50% glycerol

For applications requiring higher purity, additional purification steps such as ion-exchange or size-exclusion chromatography may be incorporated. When working with P. profundum AlaRS, consider performing certain purification steps under pressure conditions that mimic the native environment, as this may help maintain the enzyme's native conformation and activity.

This protocol is based on established methods for E. coli AlaRS purification , adapted for the piezophilic nature of P. profundum.

How can the aminoacylation activity of P. profundum AlaRS be measured in vitro?

The aminoacylation activity of P. profundum AlaRS can be measured using established radioactive assays that track the formation of aminoacyl-tRNA:

Materials Required:

• Purified P. profundum AlaRS enzyme

• In vitro transcribed tRNA^Ala or total tRNA extract

• [14C]-labeled alanine

• ATP and appropriate reaction buffers

Steady-State Aminoacylation Assay Protocol:

• Prepare a reaction mixture containing 50 nM AlaRS, 0-30 μM tRNA^Ala, 8 mM ATP in aminoacylation buffer (0.5 M HEPES [pH 7.2], 150 mM KCl, and 50 mM MgCl2)

• Add 50 μM [14C]-alanine to initiate the reaction

• Incubate at both standard (atmospheric pressure) and high pressure conditions (e.g., 28 MPa) at appropriate temperatures (15°C and 37°C)

• At regular intervals (0-4 minutes), withdraw samples and spot onto filter papers pre-soaked with 5% trichloroacetic acid (TCA)

• Wash the filters three times with 5% TCA followed by one wash with 70% ethanol

• Dry the filters and quantify radioactivity by liquid scintillation counting

• Plot the data and fit to a Michaelis-Menten curve to determine kinetic parameters

For P. profundum AlaRS, it would be particularly informative to compare kinetic parameters under different pressure conditions to assess how pressure affects enzyme activity .

What methods are available for assessing the editing activity of P. profundum AlaRS?

The editing (proofreading) activity of P. profundum AlaRS can be assessed using two complementary approaches:

1. Misaminoacylation Assay:

• Prepare a reaction containing 5 μM AlaRS, 5 μM tRNA^Ala, 8 mM ATP, and appropriate buffers

• Add non-cognate amino acids (750 μM [14C]-glycine or [3H]-serine)

• Incubate at desired temperature and pressure conditions

• At regular intervals (0-30 minutes), withdraw samples and spot onto TCA-soaked filter papers

• Process and quantify as in the aminoacylation assay

• Lower misaminoacylation rates indicate more efficient editing

2. Deacylation Assay:

• First generate misaminoacylated tRNA (Gly-tRNA^Ala or Ser-tRNA^Ala) using an editing-deficient AlaRS variant (e.g., with C666A mutation)

• Isolate the misaminoacylated tRNA

• Add wild-type P. profundum AlaRS to the pre-formed misaminoacylated tRNA

• Monitor the deacylation by quantifying the decrease in TCA-precipitable radioactivity over time

Experimental Considerations:

• Perform these assays under both atmospheric and high-pressure conditions

• Include controls with E. coli AlaRS for comparison

• For P. profundum studies, it would be valuable to assess whether pressure affects editing efficiency, particularly since accurate translation is crucial under extreme environmental conditions

How does high pressure affect the structure and function of P. profundum AlaRS?

High pressure likely induces specific structural changes in P. profundum AlaRS that affect its catalytic and editing functions. Based on proteomics studies of P. profundum under pressure, the following effects might be observed:

Structural Adaptations:

• Pressure may induce conformational changes that optimize the active site geometry for catalysis under high-pressure conditions

• Intersubunit interactions may be modified to maintain proper quaternary structure under pressure

• Hydration layers surrounding the protein may be reorganized, affecting substrate binding and product release

Functional Effects:

• Kinetic parameters (kcat, KM) may show pressure-optimized values, with optimal activity at around 28 MPa, corresponding to P. profundum's native deep-sea environment

• The balance between aminoacylation and editing activities may be pressure-dependent, potentially showing different pressure optima

• Substrate specificity and discrimination between cognate and non-cognate amino acids may be enhanced at high pressure

P. profundum has been shown to alter its proteome significantly under high-pressure conditions, with key metabolic pathways being differentially regulated . Its AlaRS may show similar pressure-dependent regulation, potentially playing a role in the organism's ability to adapt to varying pressure environments.

What experimental approaches can be used to study P. profundum AlaRS activity under high pressure?

Studying enzymatic activity under high pressure requires specialized equipment and modified protocols:

High-Pressure Experimental Systems:

• High-pressure stopped-flow devices for rapid kinetic measurements

• High-pressure vessels equipped with optical windows for spectroscopic studies

• High-pressure bioreactors for cell-based studies of AlaRS function

Methodological Approaches:

• Comparative Kinetic Analysis:

  • Perform aminoacylation and editing assays at various pressures (0.1-100 MPa)

  • Determine pressure-dependent changes in kinetic parameters

  • Generate pressure-activity profiles to identify optimal pressure conditions

• Structural Studies Under Pressure:

  • High-pressure circular dichroism to assess secondary structure changes

  • High-pressure fluorescence spectroscopy to monitor tertiary structure alterations

  • If feasible, high-pressure crystallography or NMR studies

• Molecular Dynamics Simulations:

  • Computational modeling of P. profundum AlaRS under various pressure conditions

  • Identification of key pressure-sensitive regions and residues

When designing these experiments, it's essential to include appropriate controls such as E. coli AlaRS, which is not adapted to high pressure, to differentiate between general pressure effects and specific adaptations in the P. profundum enzyme .

How do pressure-adaptive features of P. profundum AlaRS compare with other tRNA synthetases from this organism?

P. profundum likely possesses a suite of pressure-adapted tRNA synthetases, each with specific modifications for high-pressure function. Comparative analysis would reveal:

Comparative Analysis Framework:

Proteomic studies of P. profundum have shown that proteins involved in translation are differentially expressed under high-pressure conditions . Aminoacyl-tRNA synthetases, including AlaRS, likely play crucial roles in pressure adaptation by ensuring accurate protein synthesis under the organism's native high-pressure conditions.

A comprehensive comparative analysis would provide insights into common pressure-adaptive strategies across the tRNA synthetase family and enzyme-specific adaptations that reflect the particular chemistry of each amino acid charging reaction.

How can site-directed mutagenesis be used to investigate critical residues in P. profundum AlaRS?

Site-directed mutagenesis offers a powerful approach to dissect the structure-function relationships in P. profundum AlaRS, particularly for investigating pressure-adaptive features:

Mutagenesis Strategy:

• Target Selection:

  • The editing site cysteine (equivalent to E. coli C666)

  • Conserved methionine residues that might be susceptible to oxidation

  • Residues unique to piezophilic AlaRS compared to mesophilic counterparts

  • Interface residues involved in tRNA binding

• Mutation Types:

  • Conservative substitutions (e.g., Cys→Ser) to maintain chemical properties

  • Non-conservative changes (e.g., Cys→Ala) to eliminate specific functionalities

  • Introduction of residues found in non-piezophilic organisms to test pressure adaptation hypotheses

Experimental Protocol:

• Design mutagenic primers with appropriate base changes

• Perform PCR-based site-directed mutagenesis using the wild-type P. profundum alaS gene as template

• Digest template DNA with DpnI

• Transform into competent E. coli cells

• Verify mutations by sequencing

• Express and purify mutant proteins

• Characterize mutants under varying pressure conditions (0.1-100 MPa)

By comparing the activities of mutant enzymes under different pressure conditions, researchers can identify residues critical for pressure adaptation and distinguish them from those essential for basic catalytic functions .

How does oxidative stress affect P. profundum AlaRS function compared to E. coli AlaRS?

Given that deep-sea environments can differ in oxygen availability, understanding how P. profundum AlaRS responds to oxidative stress compared to its E. coli counterpart is valuable:

Comparative Analysis Framework:

E. coli AlaRS has been shown to maintain proofreading activity under oxidative stress, providing a mechanism of stress resistance . For P. profundum AlaRS, we might hypothesize:

• Oxidation Sensitivity:

  • P. profundum AlaRS may possess fewer oxidation-sensitive residues, particularly in the editing domain

  • The critical editing site cysteine might be protected by surrounding residues or by a more compact structure

• Experimental Approach:

  • Expose purified P. profundum and E. coli AlaRS to oxidative conditions (e.g., H2O2 treatment)

  • Measure aminoacylation and editing activities before and after oxidation

  • Identify oxidized residues using mass spectrometry

  • Conduct DTNB analysis to quantify accessible cysteine residues

• Expected Outcomes:

  • If P. profundum lives in environments with variable oxygen levels, its AlaRS might show enhanced resistance to oxidation

  • Alternatively, if consistently exposed to low oxygen, it might have reduced protective mechanisms

This comparative analysis would provide insights into how environmental pressures shape the evolution of stress responses in translation-related enzymes from organisms inhabiting different ecological niches .

What is the relationship between P. profundum AlaRS activity and the organism's metabolic adaptation to high pressure?

The integration of AlaRS function with broader metabolic adaptations in P. profundum reveals complex regulatory networks:

Metabolic Context:
Proteomic studies have shown that P. profundum differentially regulates key metabolic pathways under high pressure. Proteins involved in glycolysis/gluconeogenesis are up-regulated at high pressure, while several oxidative phosphorylation proteins are up-regulated at atmospheric pressure . This metabolic reconfiguration likely impacts aminoacyl-tRNA synthetase function in several ways:

• Energy Coupling:

  • AlaRS requires ATP for aminoacylation

  • Pressure-dependent changes in energy metabolism may affect ATP availability

  • The enzyme may have evolved to function optimally under the ATP concentrations typical at high pressure

• Protein Synthesis Demands:

  • Under high pressure, P. profundum may require different patterns of protein synthesis

  • AlaRS activity may be coordinated with the expression of pressure-regulated genes

  • The frequency of alanine codons in pressure-induced proteins may correlate with AlaRS activity levels

• Experimental Approaches:

  • Compare AlaRS activity with cellular ATP concentrations under different pressure conditions

  • Analyze transcriptomics/proteomics data to correlate AlaRS expression with metabolic shifts

  • Investigate whether AlaRS itself is differentially expressed under varying pressure conditions

This systems-level analysis would provide insights into how translational fidelity mechanisms are integrated with broader metabolic adaptation strategies in piezophilic organisms .

What are common challenges in working with recombinant P. profundum AlaRS and how can they be addressed?

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

Challenge 1: Protein Solubility and Stability

• Problem: AlaRS from a piezophilic organism may fold incorrectly at atmospheric pressure

• Solutions:

  • Express at lower temperatures (16-18°C)

  • Use solubility-enhancing fusion tags (SUMO, MBP)

  • Include pressure treatment during or after purification

  • Optimize buffer conditions with osmolytes that mimic high-pressure effects

Challenge 2: Activity Retention

• Problem: Loss of enzymatic activity during purification or storage

• Solutions:

  • Minimize purification steps and time

  • Include reducing agents (DTT, β-mercaptoethanol) in buffers

  • Store enzyme in high-glycerol buffers (30-50%)

  • Aliquot and flash-freeze for long-term storage

  • Consider periodic pressure treatment to maintain native conformation

Challenge 3: In vitro Transcribed tRNA Quality

• Problem: Poor quality tRNA affecting activity measurements

• Solutions:

  • Optimize in vitro transcription conditions

  • Implement rigorous tRNA purification protocols

  • Verify tRNA integrity by gel electrophoresis

  • Quantify active tRNA fraction through plateau aminoacylation

These solutions are based on established protocols for AlaRS from other organisms , with modifications to address the unique challenges of working with enzymes from piezophilic organisms.

How can researchers differentiate between pressure effects on P. profundum AlaRS activity and experimental artifacts?

Distinguishing genuine pressure effects from artifacts requires careful experimental design:

Control Measures:

• Parallel Analysis of Mesophilic AlaRS:

  • Test E. coli AlaRS under identical conditions

  • Effects observed only in P. profundum AlaRS likely represent adaptations

  • Effects common to both enzymes may be general pressure responses or artifacts

• Pressure-Cycling Experiments:

  • Subject enzyme to pressure cycles and measure activity after each cycle

  • True adaptations should show reversible responses

  • Irreversible changes may indicate denaturation or artifacts

• Concentration-Dependence Testing:

  • Pressure effects on protein-protein or protein-substrate interactions show concentration dependence

  • Artifacts often do not vary systematically with concentration

  • Perform assays at various enzyme and substrate concentrations

• Statistical Validation:

  • Conduct multiple independent experiments

  • Apply rigorous statistical analysis to distinguish significant effects

  • Consider Bayesian approaches for hypothesis testing regarding pressure effects

By implementing these controls, researchers can confidently attribute observed differences to genuine pressure adaptations in P. profundum AlaRS rather than experimental variables or artifacts .

What are the implications of P. profundum AlaRS research for understanding evolutionary adaptations to extreme environments?

Research on P. profundum AlaRS provides valuable insights into evolutionary adaptations to high-pressure environments:

Evolutionary Significance:

• Molecular Adaptation Mechanisms:

  • Identifies specific amino acid substitutions that confer pressure resistance

  • Reveals whether adaptations occur through subtle modifications or major structural changes

  • Helps distinguish between direct pressure adaptations and secondary adaptations to correlated environmental factors

• Translational Quality Control:

  • Demonstrates how fundamental processes like translation maintain accuracy under extreme conditions

  • Reveals whether error rates are modulated as part of stress response

  • May uncover novel quality control mechanisms specific to high-pressure environments

• Comparative Genomics Applications:

  • Signature analysis of AlaRS sequences from organisms inhabiting different depths

  • Identification of convergent evolutionary solutions to high-pressure adaptation

  • Development of predictive models for pressure adaptation based on sequence features

• Exobiology Relevance:

  • Insights into possible life under high-pressure conditions on other planetary bodies

  • Understanding the fundamental limits and adaptations of biological systems

  • Development of biosignature detection strategies for high-pressure environments

These fundamental insights extend beyond P. profundum to inform our broader understanding of how life adapts to extreme conditions, with applications ranging from deep-sea ecology to astrobiology .