Recombinant Photobacterium profundum 50S ribosomal protein L1 (rplA)

What is Photobacterium profundum and why is its ribosomal protein L1 important for research?

Photobacterium profundum is a deep-sea Gammaproteobacterium belonging to the family Vibrionaceae that serves as a model organism for studying adaptation to extreme environments . The bacterium was originally isolated from the Sulu Sea in 1986 and has been cultivated in several wild-type strains, including SS9, 3TCK, DJS4, and 1230 . P. profundum strain SS9 has optimal growth at 15°C and 28 MPa, qualifying it as both a psychrophile (cold-loving) and a piezophile (pressure-loving organism) .

The 50S ribosomal protein L1 (rplA) from P. profundum is of particular interest because ribosomal proteins have been identified as crucial for both low-temperature and high-pressure growth adaptation . Genome analysis has revealed that genes for ribosome assembly and function are important for adaptation to these extreme conditions . The adaptive mechanisms of ribosomal components represent a critical area of research for understanding how deep-sea organisms function under high hydrostatic pressure conditions.

How does the structure of P. profundum rplA differ from mesophilic homologs?

The structure of P. profundum rplA has evolved specific adaptations to function under high hydrostatic pressure. While maintaining the core ribosomal L1 domain structure, several key differences from mesophilic homologs include:

These structural adaptations help maintain proper protein folding and function under the high hydrostatic pressure conditions of the deep sea environment . The increased number of salt bridges and ionic interactions helps stabilize the protein structure against pressure-induced denaturation, while the compacted hydrophobic core resists water penetration at high pressures .

What expression systems are most effective for producing recombinant P. profundum rplA?

Multiple expression systems have been utilized for recombinant production of P. profundum ribosomal proteins, each with specific advantages:

For standard structural and biochemical studies, the E. coli expression system using vectors such as pET29a has proven effective, especially when expression conditions are optimized to include cold shock at 15°C during induction . For functional studies examining pressure adaptation mechanisms, expression in the baculovirus system may better preserve native structural features despite lower yields .

How should experimental conditions be modified when studying pressure effects on P. profundum rplA structure and function?

When studying the effects of pressure on P. profundum rplA, specialized experimental approaches must be employed:

• Pressure application methods:

  • For in vitro studies, custom-designed stainless steel pressure vessels capable of generating and maintaining pressures of 0.1-90 MPa are necessary .

  • Pressure perturbation should be applied incrementally (typically 10 MPa steps) to allow for structural equilibration.

  • Temperature must be precisely controlled during pressure experiments, as pressure and temperature effects are often coupled.

• Structural analysis under pressure:

  • High-pressure NMR spectroscopy using specialized pressure-resistant cells can provide atomic-level information on pressure-induced conformational changes.

  • Pressure-resistant optical cells enable fluorescence and circular dichroism measurements at elevated pressures.

  • Time-resolved studies should be conducted to capture transient intermediates during pressure adaptation.

• Functional assays:

  • RNA binding assays should be performed at relevant pressures (28 MPa for SS9 strain) to accurately assess native function .

  • Control experiments with mesophilic homologs should be included to distinguish pressure-specific adaptations.

  • Pressure cycling experiments can reveal hysteresis effects that may be biologically significant.

• Buffer considerations:

  • Buffer systems with minimal pressure dependence should be selected (e.g., phosphate buffers over Tris).

  • pH measurements must account for pressure-induced shifts (typically 0.1-0.3 pH units per 100 MPa).

  • Ionic strength should be maintained near physiological levels for the organism (~3.5% salt for marine bacteria).

These methodological considerations are essential for obtaining biologically relevant data on pressure adaptation mechanisms .

What is the role of rplA in P. profundum's adaptation to high pressure environments?

The role of rplA in P. profundum's pressure adaptation involves several molecular mechanisms:

Ribosomal proteins, including rplA, have been identified as critical for both low-temperature and high-pressure growth in P. profundum . Genomic and transcriptomic analyses have revealed that ribosome assembly and function are significantly affected by high hydrostatic pressure conditions . The adaptation mechanisms include:

Research has demonstrated that mutations affecting ribosomal components often result in pressure-sensitive phenotypes, further supporting the critical role of these proteins in high-pressure adaptation .

What biophysical techniques are most appropriate for characterizing the pressure-dependent structural changes in P. profundum rplA?

Several specialized biophysical techniques are particularly valuable for characterizing pressure-dependent structural changes in P. profundum rplA:

These techniques, when combined, provide complementary information on how rplA adapts to high pressure at the molecular level . The correlation between structural changes observed in vitro and functional adaptations in vivo should be established through careful experimental design.

How can site-directed mutagenesis be used to investigate the pressure adaptation mechanisms of P. profundum rplA?

Site-directed mutagenesis offers a powerful approach to investigating specific residues involved in pressure adaptation of P. profundum rplA:

• Selection of target residues:

  • Comparative sequence analysis between piezophilic and mesophilic homologs identifies candidate residues for mutagenesis

  • Focus on surface-exposed charged residues (potential salt bridge formers)

  • Target hydrophobic core residues that may control protein compressibility

  • Examine conserved RNA-binding residues that may affect ribosome function under pressure

• Experimental approach:

  • Generate a panel of single amino acid substitutions using standard molecular biology techniques

  • Express and purify each mutant protein using identical conditions

  • Characterize pressure stability using differential scanning calorimetry under varying pressures

  • Assess RNA binding function under normal and high-pressure conditions

  • Perform in vivo complementation studies in P. profundum pressure-sensitive mutants

• Recommended mutations to investigate:

  Mutation Type	Rationale	Expected Outcome	Analysis Method
  Charged → Neutral	Disrupt salt bridges	Decreased pressure stability	Thermal/pressure denaturation
  Hydrophobic → Smaller	Alter core packing	Changed compressibility	High-pressure SAXS
  RNA-binding → Non-binding	Affect ribosome function	Pressure-sensitive phenotype	In vivo growth assays
  Piezophile-specific → Mesophile residue	Test evolutionary adaptation	Pressure sensitivity	Comparative analysis

• Interpretation framework:

  • Mutations causing pressure sensitivity without affecting ambient pressure function indicate pressure-specific adaptations

  • Changes affecting protein stability across all pressures suggest general structural roles

  • Alterations to RNA binding that are pressure-dependent reveal functional adaptation mechanisms

This approach has been successfully employed with other pressure-adapted proteins and can be effectively applied to understand the molecular basis of rplA's role in pressure adaptation .

What are the key methodological considerations for purifying active recombinant P. profundum rplA while maintaining its native structure?

Purifying recombinant P. profundum rplA with its native structure intact requires specialized approaches:

• Expression conditions optimization:

  • Lower induction temperature (15-18°C) to match P. profundum's natural growth temperature

  • Extended expression time (16-24 hours) to allow proper folding

  • Consider expression under modest hydrostatic pressure (10-20 MPa) for optimal folding

  • Supplement growth media with osmolytes that promote proper folding (e.g., glycine betaine, proline)

• Lysis and initial purification:

  • Use gentle lysis methods (e.g., enzymatic lysis with lysozyme followed by mild sonication)

  • Include stabilizing agents in lysis buffer (10% glycerol, 1M NaCl, 5mM MgCl₂)

  • Maintain cold temperatures throughout purification (4°C)

  • Consider adding RNA fragments that bind rplA to stabilize its native conformation

• Chromatography strategy:

  Purification Step	Resin/Method	Buffer Conditions	Critical Parameters
  Initial capture	Ni-NTA affinity (for His-tagged protein)	50mM Tris pH 7.5, 500mM NaCl, 5mM MgCl₂, 10% glycerol	Slow flow rate, extended binding time
  Intermediate purification	Heparin affinity	50mM HEPES pH 7.2, 100-1000mM NaCl gradient	Mimics RNA interaction, removes misfolded species
  Polishing	Size exclusion	50mM HEPES pH 7.2, 300mM NaCl, 5mM MgCl₂, 5% glycerol	Separates aggregates and oligomers

• Structural verification methods:

  • Circular dichroism to confirm secondary structure content

  • Thermal shift assays to assess stability

  • RNA binding assays to verify functional activity

  • Limited proteolysis to evaluate correct folding

  • Negative stain electron microscopy to check for aggregation

• Storage considerations:

  • Store at high protein concentration (>1 mg/ml) to maintain stability

  • Include stabilizing agents (10% glycerol, 300mM NaCl)

  • Flash-freeze in liquid nitrogen and store at -80°C

  • Avoid repeated freeze-thaw cycles

This methodological approach has been successfully applied to other P. profundum ribosomal proteins and can be adapted specifically for rplA purification .

How can recombinant P. profundum rplA be used to study evolutionary adaptations to deep-sea environments?

Recombinant P. profundum rplA provides a valuable model for studying evolutionary adaptations to deep-sea environments through several research approaches:

• Comparative structural biology:

  • Structural comparison between rplA from P. profundum and mesophilic homologs reveals adaptations to high pressure

  • Analysis of amino acid composition can identify patterns of selection for pressure tolerance

  • Identification of specific structural elements (salt bridges, hydrophobic packing) that confer pressure resistance

• Horizontal gene transfer assessment:

  • Phylogenetic analysis of rplA sequences across marine bacteria can reveal potential horizontal gene transfer events

  • Identification of mosaic structures may indicate modular acquisition of pressure-adaptive features

  • Correlation between environmental isolation depth and sequence features can establish adaptation patterns

• Ancestral sequence reconstruction:

  • Computational reconstruction of ancestral rplA sequences allows testing of evolutionary hypotheses

  • Expression and characterization of reconstructed proteins can reveal when pressure adaptations emerged

  • Mutational pathways from mesophilic to piezophilic variants can be experimentally validated

• Experimental evolution studies:

  • Introduction of P. profundum rplA into mesophilic bacteria followed by pressure selection

  • Monitoring of compensatory mutations that occur to accommodate the piezophilic protein

  • Evaluation of fitness effects under various pressure conditions

• Ecological distribution analysis:

  • Metagenomic analysis of rplA variants across ocean depth profiles

  • Correlation of sequence features with environmental parameters (pressure, temperature, nutrients)

  • Testing for evidence of convergent evolution in diverse deep-sea lineages

This research has significant implications for understanding how life adapts to extreme environments and may provide insights into the molecular mechanisms of protein evolution under selective pressure .

What is the current understanding of how P. profundum ribosomal proteins contribute to pressure-resistant translation?

Current research indicates that P. profundum ribosomal proteins, including rplA, contribute to pressure-resistant translation through several mechanisms:

• Structural stabilization of ribosome integrity:
  P. profundum ribosomal proteins contain specific adaptations that maintain the structural integrity of the ribosome under high hydrostatic pressure . These adaptations include modified surface charge distributions, optimized salt bridge networks, and altered hydrophobic cores that resist pressure-induced denaturation.

• Pressure-resistant protein-RNA interactions:
  Research has shown that the interaction between ribosomal proteins and rRNA in piezophilic bacteria is maintained under pressure conditions that would disrupt these interactions in mesophilic organisms . This preservation of critical ribosome architecture enables continued translation function.

• Differential gene expression under pressure:
  Transcriptomic analysis has revealed that P. profundum modulates the expression of ribosomal components in response to pressure changes . This adaptive response allows the organism to maintain optimal translation machinery configuration for different pressure environments.

• Evidence from mutant studies:
  Genetic studies have demonstrated that mutations in ribosomal proteins often result in pressure-sensitive phenotypes, indicating their essential role in pressure adaptation . For example, transposon mutagenesis studies have identified numerous ribosomal protein genes as conditionally required for high-pressure growth.

• Pressure effects on translation kinetics:
  Current models suggest that adapted ribosomal proteins alter the energy landscape of translation, specifically modifying the rates of:

  Translation Step	Effect of Pressure in Mesophiles	Adaptation in P. profundum
  Initiation	Severely inhibited	Maintained rate through structural adaptations
  Elongation	Slowed	Optimized tRNA binding and translocation
  Termination	Affected by misfolding	Enhanced release factor interactions
  Ribosome recycling	Impaired	Adapted dissociation dynamics

Research continues to elucidate the precise molecular mechanisms by which these adaptations enable pressure-resistant translation, with ribosomal proteins playing a central role in this process .

How can functional assays be designed to evaluate the pressure-dependent activity of recombinant P. profundum rplA?

To evaluate the pressure-dependent activity of recombinant P. profundum rplA, specialized functional assays must be designed that incorporate both high-pressure conditions and relevant biological activities:

• RNA binding assays under pressure:

  • Filter binding assays conducted in pressure-resistant chambers

  • Fluorescence anisotropy measurements with labeled RNA substrates under varying pressures

  • Surface plasmon resonance adapted for high-pressure applications

  • Assessment of binding kinetics (kon and koff) as functions of pressure

• Reconstituted translation systems:

  • In vitro translation assays using purified components (PURE system approach)

  • Incorporation of recombinant P. profundum rplA into hybrid ribosomes

  • Measurement of translation rates and accuracy under pressure gradient (0.1-50 MPa)

  • Comparison with systems containing mesophilic rplA variants

• Structural stability assessments:

  • Hydrogen-deuterium exchange mass spectrometry under varying pressures

  • Pressure-dependent circular dichroism spectroscopy

  • Differential scanning calorimetry with pressure as the variable

  • Correlation of stability parameters with functional activity

• Experimental design considerations:

  Parameter	Range to Test	Equipment Requirements	Data Analysis Approach
  Pressure	0.1-50 MPa (focus on 28 MPa)	Custom high-pressure chambers	Pressure-dependent kinetic modeling
  Temperature	4-25°C (focus on 15°C)	Precise temperature control within pressure chamber	Arrhenius plots at different pressures
  Salt concentration	0.3-0.5 M NaCl	Samples prepared at relevant ionic strength	Evaluate electrostatic contribution to pressure adaptation
  Mg²⁺ concentration	5-20 mM	Critical for RNA structure stabilization	Assess divalent ion dependence under pressure

• Complementation studies:

  • Introduction of recombinant rplA variants into P. profundum strains with rplA mutations

  • Assessment of pressure tolerance restoration

  • Growth rate measurements under pressure gradients

  • Correlation of in vitro biochemical properties with in vivo phenotypes

These assays should be designed with appropriate controls, including parallel experiments with rplA from mesophilic organisms to highlight pressure-specific adaptations .

What insights can structural studies of P. profundum rplA provide about the evolution of extremophilic adaptations?

Structural studies of P. profundum rplA can provide significant insights into the evolution of extremophilic adaptations through multiple analytical approaches:

• Comparative structural analysis across depth gradients:

  • Structures of rplA homologs from bacteria isolated at different ocean depths can reveal progressive adaptations

  • Identification of convergently evolved features in unrelated deep-sea lineages

  • Mapping of adaptive mutations onto structural landscapes to identify "hotspots" for pressure adaptation

• Structure-function correlations:

  • Integration of structural data with functional measurements under pressure

  • Identification of critical residues that maintain function under extreme conditions

  • Mechanistic understanding of how specific structural elements contribute to pressure resistance

• Evolutionary insights from structural plasticity:

  • Analysis of protein dynamics rather than static structures

  • Characterization of conformational energy landscapes under varying pressures

  • Understanding how proteins evolve alternative energy minima to function across pressure ranges

• Adaptation mechanisms revealed through structural studies:

  Structural Feature	Evolutionary Mechanism	Detection Method	Significance
  Surface electrostatics	Selection for increased negative charge	Electrostatic potential mapping	Stabilizes water interactions under pressure
  Internal cavities	Reduction in void volume	Cavity analysis algorithms	Minimizes pressure-induced water penetration
  Protein flexibility	Modulation of dynamic properties	NMR relaxation measurements	Balances stability with necessary conformational changes
  Secondary structure propensity	Selection for pressure-resistant structural elements	Hydrogen-deuterium exchange	Identifies structurally conserved regions important for function

• Insights into general principles of protein adaptation:

  • Determination whether extremophilic adaptations follow consistent patterns or multiple solutions

  • Assessment if adaptations are achieved through gradual change or punctuated evolution

  • Evaluation whether structural adaptation comes with functional trade-offs

These structural studies not only illuminate the specific adaptations of P. profundum rplA but also contribute to our broader understanding of how proteins evolve to function in extreme environments, which has implications for astrobiology, biotechnology, and evolutionary biology .

What are the common challenges in expressing and purifying functional P. profundum ribosomal proteins and how can they be overcome?

Researchers face several challenges when expressing and purifying functional P. profundum ribosomal proteins, with specific solutions for each issue:

• Protein solubility issues:

  • Challenge: Ribosomal proteins often form inclusion bodies when overexpressed in E. coli.

  • Solution: Lower induction temperature (15-18°C), reduce IPTG concentration (0.1-0.2 mM), co-express with chaperones (GroEL/ES, DnaK/J), or use solubility-enhancing fusion tags (SUMO, MBP) .

• Maintaining native structure:

  • Challenge: Proteins adapted to high pressure may not fold correctly under atmospheric pressure.

  • Solution: Express under mild pressure conditions (10-20 MPa) using specialized equipment, include osmolytes (trimethylamine N-oxide, betaine) in growth media, and maintain physiologically relevant salt concentrations (300-500 mM NaCl) .

• Protein stability during purification:

  • Challenge: Ribosomal proteins are prone to degradation and aggregation during purification.

  • Solution: Work rapidly at 4°C, include protease inhibitors, maintain high ionic strength buffers, add stabilizing agents (glycerol, arginine), and consider one-step purification methods to minimize handling time .

• RNA contamination:

  • Challenge: Ribosomal proteins often co-purify with cellular RNA due to their natural binding affinity.

  • Solution: Include RNase treatment steps, perform heparin affinity chromatography, use high-salt washes (1-2 M NaCl), and implement polyethyleneimine precipitation to remove nucleic acids .

• Functional verification:

  • Challenge: Confirming that purified proteins retain native function is difficult without assembled ribosomes.

  • Solution: Develop specific RNA binding assays, perform thermal/pressure stability tests, use circular dichroism to verify secondary structure, and compare with mesophilic homologs as benchmarks.

• Purification troubleshooting guide:

  Problem	Possible Causes	Diagnostic Test	Solution
  Low yield	Poor expression, degradation	SDS-PAGE of whole cells vs. purified fraction	Optimize induction, add protease inhibitors
  Aggregation	Improper folding, concentration too high	Dynamic light scattering	Add stabilizing agents, purify under milder conditions
  Co-purifying contaminants	RNA binding, protein-protein interactions	260/280 nm absorbance ratio	Additional purification steps, nuclease treatment
  Loss of activity	Denaturation, critical cofactor loss	Functional assays	Include Mg²⁺ and K⁺ ions, stabilize with RNA fragments
  Precipitation during storage	Buffer incompatibility, freeze-thaw damage	Visual inspection, centrifugation test	Optimize storage buffer, add cryoprotectants

• Expression system selection:

  • Challenge: Standard expression systems may not produce properly folded psychrophilic/piezophilic proteins.

  • Solution: Test multiple expression systems (E. coli Arctic Express, psychrophilic bacterial hosts, cell-free systems) and optimize for the specific protein .

These approaches have been successfully applied to other P. profundum proteins and can be adapted specifically for ribosomal proteins including rplA .

How can researchers accurately simulate deep-sea pressure conditions in laboratory settings for studying P. profundum ribosomal protein function?

Accurately simulating deep-sea pressure conditions in laboratory settings requires specialized equipment and methodological considerations:

• High-pressure equipment options:

  • Hydrostatic pressure vessels: Custom-designed stainless steel pressure vessels capable of generating and maintaining pressures up to 100 MPa are the most common approach . These can be connected to hydraulic pumps for pressure control.

  • Diamond anvil cells: For microscopic samples and spectroscopic studies, diamond anvil cells can generate even higher pressures with optical access.

  • High-pressure stopped-flow devices: For kinetic measurements of fast reactions under pressure.

  • Pressure-resistant optical cells: For spectroscopic measurements while maintaining pressure.

• Experimental design considerations:

  • Temperature control: Precise temperature regulation is critical as pressure and temperature effects are often coupled. Water-jacketed pressure vessels connected to circulating water baths can maintain temperatures within ±0.1°C .

  • Pressure transmission: Pressure must be transmitted uniformly to samples. This is typically achieved using water or hydraulic fluid separated from samples by flexible barriers.

  • Sample containers: Samples should be in pressure-resistant, non-compressible containers that don't introduce artifacts. Polyethylene tubes have proven effective for biological samples .

• Pressure application protocols:

  Parameter	Recommendation	Rationale	Implementation
  Pressure range	0.1-50 MPa with 5-10 MPa increments	Covers atmospheric to deep-sea trenches	Calibrated pressure gauges with 0.1 MPa precision
  Rate of pressurization	10 MPa/min	Avoids shock effects on biological systems	Controlled hydraulic pump or manual valve systems
  Equilibration time	Minimum 30 min at each pressure	Allows system to reach steady state	Timed protocols with pressure monitoring
  Decompression rate	5-10 MPa/min	Prevents rapid pressure release effects	Controlled valve systems with flow restrictors

• Validation approaches:

  • Physical validation: Use pressure-sensitive dyes or materials with known pressure responses to verify conditions.

  • Biological validation: Include control organisms with well-characterized pressure responses (e.g., E. coli).

  • Chemical validation: Monitor pressure-dependent chemical reactions with known rate constants.

• Integrated systems for function studies:

  • For RNA binding studies: Fluorescence detection systems integrated with pressure vessels.

  • For translation assays: Specialized high-pressure chambers adapted for enzyme activity measurements.

  • For structural studies: Pressure vessels compatible with spectroscopic techniques (CD, fluorescence).

• Data analysis considerations:

  • Pressure effects should be analyzed in terms of volume changes using appropriate thermodynamic frameworks.

  • Control experiments at atmospheric pressure should always be included.

  • Reversibility should be verified by testing function after pressure release.

These approaches have been successfully employed in studies of P. profundum and other piezophilic organisms, enabling accurate simulation of deep-sea conditions in laboratory settings .

What bioinformatic approaches are most useful for analyzing the evolutionary adaptations in P. profundum ribosomal proteins?

Several specialized bioinformatic approaches are particularly valuable for analyzing evolutionary adaptations in P. profundum ribosomal proteins:

• Comparative sequence analysis:

  • Homology identification: Collect homologous sequences across bacteria from various depth environments using BLAST, HMMER and profile searches.

  • Multiple sequence alignment: Use structure-aware alignment tools (PROMALS3D, T-Coffee) that incorporate structural information for more accurate alignments of ribosomal proteins.

  • Conservation pattern analysis: Identify conserved positions within piezophiles that differ from mesophiles using ConSurf or Rate4Site algorithms.

• Phylogenetic methods:

  • Bayesian phylogenetic reconstruction: Build robust phylogenetic trees using MrBayes or BEAST with appropriate evolutionary models.

  • Ancestral sequence reconstruction: Infer ancestral sequences at each node to trace evolutionary trajectories using FastML or PAML.

  • Selection pressure analysis: Calculate dN/dS ratios to identify positions under positive selection using PAML, HyPhy, or MEME.

• Structural bioinformatics approaches:

  Approach	Tools	Application to rplA	Insights Gained
  Homology modeling	SWISS-MODEL, Phyre2	Create models of P. profundum rplA based on solved structures	Structural differences from mesophilic homologs
  Molecular dynamics	GROMACS, NAMD	Simulate protein behavior under pressure	Conformational stability and water interactions
  Electrostatic analysis	APBS, DelPhi	Map surface charge distribution	Identification of stabilizing salt bridges
  Cavity analysis	CASTp, POVME	Identify and measure internal cavities	Volume changes that affect pressure sensitivity
  Normal mode analysis	ProDy, ElNemo	Predict intrinsic flexibility	Differences in dynamic properties related to function

• Coevolutionary analysis:

  • Direct coupling analysis (DCA) and related methods to identify co-evolving residue networks within rplA.

  • Integration with structural data to identify networks important for pressure adaptation.

  • Comparison of co-evolutionary patterns between piezophilic and mesophilic lineages.

• Genomic context analysis:

  • Examine operon structure and gene neighborhood conservation across species to identify functional associations.

  • Analyze upstream regulatory regions for pressure-responsive elements using comparative genomics.

  • Investigate horizontal gene transfer patterns in ribosomal protein genes using reconciliation methods.

• Machine learning approaches:

  • Develop classifiers to predict pressure adaptation based on sequence features.

  • Use feature importance methods to identify key residues contributing to piezophilic adaptation.

  • Apply unsupervised learning to cluster sequences according to pressure adaptation patterns.