Recombinant Photobacterium profundum Cysteine--tRNA ligase 1 (cysS1)

What is Photobacterium profundum Cysteine--tRNA ligase 1 and what is its fundamental function?

Photobacterium profundum Cysteine--tRNA ligase 1 (cysS1) is an aminoacyl-tRNA synthetase that catalyzes the essential reaction attaching cysteine to its cognate tRNA molecule. The enzyme specifically catalyzes the following reaction:

ATP + L-cysteine + tRNACys → AMP + diphosphate + L-cysteinyl-tRNACys

This reaction is critical for protein synthesis as it enables the incorporation of cysteine amino acids into growing polypeptide chains during translation. P. profundum is a piezophilic (pressure-loving) bacterium that grows optimally at 28 MPa and 15°C, and its enzymes, including cysS1, have likely evolved specific adaptations to function effectively under high-pressure deep-sea conditions .

What is the optimal protocol for expressing and purifying active recombinant P. profundum cysS1?

Expression and purification of active recombinant P. profundum cysS1 requires careful attention to the piezophilic nature of the source organism. A comprehensive protocol would include:

Expression System:

• Host: E. coli BL21(DE3) or similar expression strain

• Vector: pET-based with temperature-inducible promoter

• Growth conditions: Lower temperatures (15-17°C) to mimic natural environment

• Induction: Low IPTG concentration (0.1-0.5 mM) for extended periods (16-24 hours)

Purification Strategy:

• Cell lysis under reducing conditions to prevent oxidation of cysteine residues

• Initial capture using affinity chromatography (His-tag or similar)

• Ion exchange chromatography to remove contaminants

• Size exclusion chromatography to ensure monomeric state and remove aggregates

Buffer Composition:

• Base buffer: 50 mM Tris-HCl or HEPES (pH 7.5)

• Salt: 100-300 mM NaCl or KCl

• Reducing agent: 1-5 mM DTT or β-mercaptoethanol

• Stabilizers: 10-20% glycerol and potentially osmolytes that mimic pressure effects

Throughout purification, it's essential to monitor enzyme activity using aminoacylation assays to ensure the recovered protein maintains functionality. Storage should be at -80°C in buffer containing glycerol to preserve activity for long-term use.

How should aminoacylation assays be designed to accurately measure P. profundum cysS1 activity?

Accurate measurement of P. profundum cysS1 aminoacylation activity requires careful experimental design considering both the reaction chemistry and the enzyme's piezophilic origin:

Standard Reaction Components:

• Enzyme: Purified recombinant P. profundum cysS1 (10-100 nM)

• Substrates: ATP (1-5 mM), L-cysteine (0.1-1 mM), tRNACys (0.1-10 μM)

• Buffer: 50 mM Tris or HEPES (pH 7.5), 12 mM MgCl2, 25 mM KCl

• Additives: 1 mg/mL BSA, 0.5 mM spermine, 1 mM DTT

Analytical Methods:

• Radiometric assay: Using [13C4,15N] cysteine, followed by TCA precipitation and ammonia release as described in previous studies

• HPLC separation of charged vs. uncharged tRNAs

• ATP-PPi exchange assay to measure the reverse reaction

• Mass spectrometry to detect mass increase of charged tRNA

Controls and Variables:

• Negative controls: No-enzyme, no-ATP, no-tRNA, and heat-inactivated enzyme

• Positive control: Well-characterized cysteine-tRNA ligase (e.g., E. coli)

• Temperature range: 4-37°C, with emphasis on 15°C (optimal for P. profundum)

• Pressure conditions: If available, conduct assays at both atmospheric (0.1 MPa) and elevated pressure (28 MPa)

Data Analysis:

• Initial velocity measurements to determine steady-state kinetic parameters

• Michaelis-Menten analysis to derive Km, Vmax, and kcat values

• Analysis of pressure and temperature effects on catalytic efficiency (kcat/Km)

(Note: The table above provides a framework for recording experimental results; actual values would be determined experimentally.)

How does hydrostatic pressure affect the catalytic efficiency and structure of P. profundum cysS1?

The effect of hydrostatic pressure on P. profundum cysS1 is of particular interest given the piezophilic nature of its source organism. Understanding these effects requires multi-faceted experimental approaches:

P. profundum grows optimally at 28 MPa pressure and 15°C, suggesting its cellular machinery, including cysS1, has evolved to function effectively under these conditions . Pressure can affect enzymes through several mechanisms:

• Conformational effects: High pressure can stabilize certain protein conformations while destabilizing others

• Volume changes: Reactions with negative activation volumes are accelerated by pressure

• Hydration effects: Pressure can alter the hydration shell around proteins and substrates

• Substrate binding: Pressure may modify Km values by affecting enzyme-substrate interactions

For P. profundum cysS1, pressure effects might include:

• Potential decrease in Km values at elevated pressure (28 MPa), indicating improved substrate binding

• Possible alteration of rate-limiting step at different pressures

• Structural stabilization at the organism's optimal pressure

A comprehensive experimental design would involve measuring aminoacylation kinetics across a pressure range (0.1-50 MPa) at various temperatures, particularly focusing on 15°C, the optimal growth temperature of P. profundum . High-pressure spectroscopic studies (fluorescence, circular dichroism) could provide insights into structural changes under pressure.

Comparative analysis with cysteine-tRNA ligases from non-piezophilic organisms would highlight pressure-specific adaptations in P. profundum cysS1.

What experimental approaches can distinguish between general pressure effects and adaptive features in P. profundum cysS1?

Distinguishing adaptive features from general pressure effects requires carefully designed experiments:

Comparative Enzyme Analysis:

• Compare P. profundum cysS1 with homologous enzymes from:

  • Mesophilic, atmospheric pressure organisms (e.g., E. coli)

  • Related Photobacterium species from different depth environments

  • Other piezophilic organisms from similar depths

Chimeric Protein Approach:

• Create domain-swapped chimeras between P. profundum cysS1 and mesophilic homologs

• Test chimeras for pressure response to identify domains responsible for pressure adaptation

Site-Directed Mutagenesis:

• Target residues unique to P. profundum cysS1 or conserved among piezophilic homologs

• Create "mesophilic-like" mutations and assess pressure sensitivity

• Sample experimental design based on approaches used for other aminoacyl-tRNA synthetases :

Pressure-Jump Kinetics:

• Use rapid pressure change systems to measure transient kinetic parameters

• Analyze reaction progress curves to identify pressure-sensitive steps in the catalytic cycle

Molecular Dynamics Simulations:

• Perform comparative simulations of P. profundum cysS1 and mesophilic homologs

• Analyze protein flexibility, solvent accessibility, and active site geometry at different pressures

These approaches collectively would reveal whether P. profundum cysS1's response to pressure represents adaptation or is simply a physicochemical consequence of pressure on protein structure and function.

How can site-directed mutagenesis be used to investigate critical residues in P. profundum cysS1?

Site-directed mutagenesis provides powerful insights into structure-function relationships in P. profundum cysS1. An effective research strategy would include:

Target Identification:

• Conserved catalytic residues based on homology with characterized cysteine-tRNA ligases

• Residues unique to piezophilic cysteine-tRNA ligases (identified through sequence alignment)

• Key structural elements in the three functional domains: ATP binding, cysteine recognition, and tRNA binding

Mutation Types:

• Alanine scanning of active site and substrate binding regions

• Conservative substitutions (e.g., Lys→Arg, Asp→Glu) to test specific chemical requirements

• Non-conservative substitutions to drastically alter properties

• Introduction of residues found in mesophilic homologs to test pressure adaptation hypotheses

Functional Characterization:

• Aminoacylation assays under varied pressure conditions (0.1-50 MPa)

• Substrate binding assays to separate effects on binding vs. catalysis

• Thermal and pressure stability measurements

• ATP-PPi exchange assays to measure isolated activation step

Based on studies of aminoacyl-tRNA synthetases, particularly promising targets would include :

• Conserved lysine residues in the ATP binding pocket (similar to Lys114 in yeast Trl1)

• Motif I (typically KMSKS or variants) involved in ATP binding

• Cysteine recognition residues in the amino acid binding pocket

• tRNA recognition elements (often in C-terminal domain)

A systematic mutagenesis approach would involve creating series of mutants, expressing and purifying them under identical conditions, then comparing their kinetic parameters and pressure responses to wild-type enzyme.

What molecular mechanisms might explain pressure adaptation in P. profundum cysS1?

Understanding the molecular mechanisms of pressure adaptation in P. profundum cysS1 requires consideration of several potential structural and functional adaptations:

Volume Change Optimization:

• Reactions with negative activation volumes are favored at high pressure

• P. profundum cysS1 may have evolved a catalytic mechanism with more negative ΔV‡ for the rate-limiting step

• Key structural features might include:

  • Reduced cavity volumes in the protein interior

  • More efficient packing of side chains

  • Optimization of water-protein interactions

Conformational Flexibility:
Piezophilic enzymes often display increased flexibility at high pressure compared to mesophilic homologs . For P. profundum cysS1, this might involve:

• Strategic placement of glycine residues in hinge regions

• Reduced number of rigid structural elements like proline residues

• Altered surface charge distribution to enhance hydration

Substrate Binding Adaptations:

• Modified binding pockets that undergo less compaction under pressure

• Altered electrostatic interactions that are less sensitive to pressure-induced solvent effects

• Optimized induced-fit mechanisms that function efficiently at high pressure

Comparative Data Analysis:
By studying the aminoacylation proteomic analysis of P. profundum under different pressure conditions, we can identify patterns similar to other pressure-regulated proteins :

Experimental approaches combining site-directed mutagenesis, biophysical characterization, and computational modeling would be needed to determine which of these mechanisms predominate in P. profundum cysS1.

How can contradictory kinetic data for P. profundum cysS1 be resolved through improved experimental design?

Contradictory kinetic data for P. profundum cysS1 can arise from multiple sources, including variation in experimental conditions, enzyme preparation differences, and methodological inconsistencies. A systematic approach to resolve such contradictions would include:

Standardization of Experimental Conditions:

• Precise buffer composition control: pH, ionic strength, and metal ion concentrations

• Stringent enzyme quality criteria: purity, specific activity, and absence of inhibitors

• Consistent substrate preparation: tRNA refolding, amino acid and ATP purity

• Temperature control with ±0.1°C precision

• Pressure control with calibrated equipment when applicable

Multi-Method Validation:
When contradictory results appear, employ multiple independent assay techniques:

• Direct aminoacylation assays (radiometric)

• ATP consumption measurements (coupled enzyme assays)

• Pyrophosphate release detection

• Mass spectrometry to confirm product formation

Control Experiments:

• Measure enzyme stability under assay conditions (pre-incubation tests)

• Test for product inhibition effects

• Examine time-course to ensure initial velocity conditions

• Check for aggregation state changes during the reaction

Statistical Analysis Framework:

• Implement global fitting of datasets across multiple conditions

• Apply statistical tests appropriate for enzyme kinetics (e.g., extra sum-of-squares F test)

• Establish confidence intervals for all derived parameters

• Use Bayesian approaches for complex mechanisms

Specific Troubleshooting for P. profundum cysS1:
Based on challenges observed with other aminoacyl-tRNA synthetases and pressure-adapted enzymes :

• Test for pressure-dependent conformational changes that might cause hysteresis

• Examine temperature-pressure interaction effects on kinetic parameters

• Consider product release as a potential rate-limiting step affected by pressure

• Evaluate tRNA charging efficiency separately from amino acid activation

By implementing this systematic approach, researchers can identify the source of contradictions and develop a more accurate kinetic model for P. profundum cysS1 activity.

What computational approaches can predict structure-function relationships in P. profundum cysS1 under varying pressure conditions?

Computational approaches offer powerful tools for predicting how pressure affects P. profundum cysS1 structure and function, especially when experimental high-pressure studies are challenging. Effective computational strategies include:

Homology Modeling and Structural Analysis:

• Build a 3D model of P. profundum cysS1 based on known structures of cysteine-tRNA ligases

• Analyze structural features potentially associated with pressure adaptation:

  • Cavity volumes and packing density

  • Surface charge distribution

  • Flexibility of catalytic regions

  • Solvent-accessible surface area

Molecular Dynamics Simulations:

• Perform pressure-explicit MD simulations at various pressures (0.1-100 MPa)

• Analyze:

  • Protein compressibility and volume fluctuations

  • Conformational changes in active site geometry

  • Water penetration and protein hydration

  • Dynamics of substrate binding regions

Quantum Mechanics/Molecular Mechanics (QM/MM):

• Model the chemical reaction mechanism under different pressure conditions

• Calculate activation energies and transition state structures

• Predict pressure effects on rate-limiting steps

Sequence-Based Approaches:

• Comparative analysis of cysteine-tRNA ligases from organisms adapted to different pressure environments

• Identification of pressure-adaptive signatures through statistical coupling analysis

• Prediction of pressure-sensitive regions through conservation analysis

Integrated Computational-Experimental Workflow:

These computational approaches provide hypotheses that can guide experimental design and help interpret experimental results in the context of molecular mechanisms of pressure adaptation.

How does P. profundum cysS1 compare functionally to cysteine-tRNA ligases from non-piezophilic organisms?

Comparative analysis between P. profundum cysS1 and cysteine-tRNA ligases from non-piezophilic organisms reveals important adaptations and conserved features:

Conserved Catalytic Mechanism:
All cysteine-tRNA ligases, including P. profundum cysS1, catalyze the same fundamental reaction: ATP + L-cysteine + tRNACys → AMP + diphosphate + L-cysteinyl-tRNACys . This reaction involves:

• Activation of cysteine with ATP to form cysteinyl-AMP

• Transfer of cysteine to the 3' end of tRNACys

• Release of charged tRNA and AMP

Expected Functional Differences:
Based on studies of other pressure-adapted enzymes in P. profundum and studies of other aminoacyl-tRNA synthetases , P. profundum cysS1 likely exhibits:

• Pressure-optimized kinetic parameters:

  • Potentially lower Km values at high pressure (28 MPa)

  • Higher catalytic efficiency (kcat/Km) at elevated pressure

  • Different rate-limiting step compared to mesophilic homologs

• Temperature adaptations:

  • Higher activity at lower temperatures (10-15°C)

  • Potentially broader temperature range of activity

  • Different thermodynamic parameters (ΔH‡, ΔS‡)

• Structural adaptations:

  • Modified flexibility in key catalytic regions

  • Altered substrate binding dynamics

  • Potentially different oligomeric state stability

Comparative Experimental Approaches:
To systematically compare P. profundum cysS1 with non-piezophilic homologs:

• Express and purify both enzymes under identical conditions

• Measure aminoacylation kinetics across pressure (0.1-50 MPa) and temperature (4-37°C) ranges

• Determine substrate specificity and fidelity under various conditions

• Analyze stability and folding properties

These comparisons would reveal whether P. profundum cysS1 represents a specialized adaptation to high-pressure environments or simply functions with different optimal conditions than its mesophilic counterparts.

What are the most promising research directions for understanding P. profundum cysS1 structure-function relationships?

Future research on P. profundum cysS1 should focus on several key directions to advance our understanding of this pressure-adapted enzyme:

Structural Determination:

• X-ray crystallography or cryo-EM studies of P. profundum cysS1 in different functional states

• Comparative structural analysis with mesophilic homologs

• High-pressure structural studies using specialized equipment

Mechanistic Investigations:

• Pre-steady-state kinetics to identify rate-limiting steps under various pressure conditions

• Isotope effect studies to probe chemical mechanism

• Single-molecule studies to examine conformational dynamics

Evolutionary Perspectives:

• Comparative analysis of cysS1 genes across Photobacterium species from different depths

• Reconstructed evolutionary trajectories to identify key adaptive mutations

• Horizontal gene transfer analysis to understand the acquisition of pressure adaptation

Biotechnological Applications:

• Engineering pressure adaptation into mesophilic enzymes based on insights from P. profundum cysS1

• Developing P. profundum cysS1 as a model system for understanding pressure effects on protein function

• Exploring potential applications in high-pressure biocatalysis

By pursuing these research directions, scientists can gain deeper insights into how enzymes adapt to extreme environments and potentially develop new tools for both fundamental research and biotechnological applications.