Recombinant Photobacterium profundum Methionine import ATP-binding protein MetN (metN)

What is the Photobacterium profundum MetN protein and its primary function?

The MetN protein in Photobacterium profundum is the ATP-binding component (nucleotide-binding domain) of the methionine ABC transporter system. It functions as part of a complex that typically includes a transmembrane domain (MetI) and a substrate-binding protein (MetQ). This transport system is responsible for the ATP-dependent import of methionine and its derivatives across the cell membrane. Similar to the E. coli MetNI system, the P. profundum MetN protein likely exhibits ATPase activity that drives conformational changes in the transporter complex, facilitating substrate translocation . The protein belongs to the broader ABC transporter superfamily, which utilizes ATP hydrolysis as an energy source for active transport of substrates.

How does the genetic organization of metN differ in P. profundum compared to other bacteria?

In Photobacterium profundum SS9, the metN gene is part of the genome that has been extensively studied using genetic, genomic, and functional genomic approaches . While the specific organization of the metN gene locus in P. profundum is not explicitly detailed in the provided search results, it likely follows patterns similar to other bacterial methionine transport systems. Based on comparative genomics, the metN gene in P. profundum would be expected to be part of an operon that includes genes encoding other components of the methionine transport system. The genomic context may include regulatory elements that respond to methionine availability and environmental conditions, particularly pressure, which is a significant factor for deep-sea bacteria like P. profundum SS9.

What are the key structural domains of the P. profundum MetN protein?

The P. profundum MetN protein likely contains standard structural features of nucleotide-binding domains (NBDs) found in ABC transporters, including:

• Walker A motif (P-loop): A conserved sequence involved in ATP binding

• Walker B motif: Essential for ATP hydrolysis

• ABC signature motif (C-loop): Unique to ABC transporters

• Q-loop and H-loop: Involved in coordinating the nucleotide and communicating between domains

• D-loop: Involved in the interface between the two NBDs

Additionally, by analogy with the E. coli MetNI transporter, the P. profundum MetN may contain a C-terminal regulatory domain that functions in trans-inhibition, regulating transport activity in response to intracellular methionine concentrations . This structural organization enables the protein to couple ATP hydrolysis with substrate transport while also responding to cellular methionine levels.

What are the optimal conditions for recombinant expression of P. profundum MetN?

For recombinant expression of P. profundum MetN, researchers should consider the following methodological approach:

• Expression System Selection: E. coli BL21(DE3) or similar strains are typically suitable for expressing bacterial membrane-associated proteins. For challenging expressions, specialized strains like C41(DE3) or C43(DE3) may offer advantages.

• Vector Design: Use a vector containing an inducible promoter (T7 or tac) and include affinity tags (His6, FLAG, or Strep) for purification. The pET or pBAD vector systems are commonly used.

• Temperature Optimization: Deep-sea proteins often express better at lower temperatures. Initial expression tests should include 15°C, 20°C, and 25°C conditions to determine optimal expression temperature.

• Induction Parameters: For IPTG-inducible systems, use concentrations between 0.1-0.5 mM; higher concentrations may lead to inclusion body formation. Auto-induction media can also be considered for gentler expression.

• Pressure Considerations: Since P. profundum is a piezophile adapted to high-pressure environments (optimally at 30 MPa) , expression under modest pressure (10-15 MPa) using specialized equipment may improve folding of pressure-adapted proteins.

The expression methodology should be validated by SDS-PAGE and Western blot analysis to confirm successful production of the target protein.

What purification strategies are most effective for obtaining high-purity recombinant P. profundum MetN?

A multi-step purification strategy is recommended for isolating high-purity P. profundum MetN:

• Cell Lysis: Use a combination of enzymatic (lysozyme) and mechanical (sonication or high-pressure homogenization) methods in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors.

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resin for His-tagged protein. Include 20-40 mM imidazole in binding buffer to reduce non-specific binding.

• Intermediate Purification: Ion exchange chromatography can be used as a second step, with the specific resin (anion or cation exchange) selected based on the protein's theoretical isoelectric point.

• Polishing Step: Size exclusion chromatography using Superdex 200 or similar matrix in buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl₂, and 1 mM DTT.

• Quality Assessment: Analyze purity by SDS-PAGE (>95% purity target), verify identity by mass spectrometry, and assess functional activity through ATPase assays.

For protein intended for structural studies, additional steps like tag removal and buffer optimization may be necessary. The inclusion of detergents (0.02-0.05% DDM or 0.5% CHAPS) during purification may improve yield and stability if the protein associates with membrane components.

How can researchers assess the functional activity of purified recombinant P. profundum MetN?

Functional assessment of purified P. profundum MetN should include multiple complementary approaches:

• ATPase Activity Assay: Measure ATP hydrolysis using either:

  • Malachite green phosphate detection assay

  • Coupled enzyme assay with pyruvate kinase and lactate dehydrogenase

  • [γ-³²P]ATP-based assay for highly sensitive detection

• Nucleotide Binding Analysis:

  • Fluorescence-based assays using TNP-ATP or MANT-ATP

  • Isothermal titration calorimetry (ITC) to determine binding constants

  • Surface plasmon resonance (SPR) for binding kinetics

• Functional Reconstitution:

  • Reconstitute with MetI and MetQ components in liposomes

  • Measure transport of ¹⁴C or ³H-labeled methionine into liposomes

  • Assess transport under varying pressure conditions

• Thermal Stability Assessment:

  • Differential scanning fluorimetry (DSF) to monitor protein stability

  • Circular dichroism (CD) spectroscopy for secondary structure analysis

A sample data table showing expected ATPase activity parameters:

Note: These values are hypothetical and should be experimentally determined for P. profundum MetN, as actual values may differ based on specific conditions and protein preparation methods.

How can researchers investigate the pressure-dependent activity of P. profundum MetN?

Investigating pressure-dependent activity of P. profundum MetN requires specialized equipment and methodological considerations:

• High-Pressure Enzymatic Assays:

  • Use pressure vessels equipped with optical windows for spectrophotometric assays

  • Employ pressure-resistant sealed reaction chambers with rapid decompression capabilities for sampling

  • Utilize HPDS (high-pressure deep-sea cell) systems similar to those described for motility studies

• Experimental Design:

  • Establish a pressure gradient series (0.1 MPa, 10 MPa, 30 MPa, 50 MPa, 100 MPa)

  • Include appropriate controls: atmospheric pressure samples, pressure-sensitive proteins (e.g., from E. coli), and other P. profundum proteins

  • Measure activity immediately after pressurization and following pressure release to assess reversibility

• Data Analysis Framework:

  • Calculate pressure activation volumes (ΔV‡) from rate constants at different pressures

  • Determine pressure midpoint (Pm) values where activity is 50% of maximum

  • Plot activity profiles as function of pressure to identify optimal conditions

• Structural Analysis Under Pressure:

  • Small-angle X-ray scattering (SAXS) under pressure to monitor conformational changes

  • High-pressure NMR studies for detailed structural information

  • Molecular dynamics simulations to predict pressure effects on protein dynamics

The experimental approach should be modeled after methods used for studying other pressure-adapted proteins from P. profundum SS9, which has been extensively characterized for growth at pressures ranging from atmospheric to 90 MPa, with optimal growth occurring at approximately 28 MPa .

What methodologies are available for investigating the substrate specificity of P. profundum MetN?

Investigating substrate specificity of P. profundum MetN requires a multi-faceted approach:

• Competitive Binding Assays:

  • Radiolabeled methionine displacement assays with various methionine derivatives

  • Fluorescence-based competition assays using fluorescent methionine analogs

  • Surface plasmon resonance (SPR) competition studies

• Transport Assays in Reconstituted Systems:

  • Liposome-based transport assays with different substrates

  • Whole-cell uptake assays using recombinant expression systems

  • Isothermal titration calorimetry to measure binding energetics

• Substrate-Dependent ATPase Stimulation:

  • Compare ATPase activity in the presence of different methionine derivatives

  • Generate substrate concentration-dependent activity curves

  • Based on E. coli MetNI data, evaluate both L-methionine and D-methionine derivatives, including selenomethionine

• Structural Biology Approaches:

  • Co-crystallization with different substrates

  • Molecular docking simulations

  • HDX-MS (hydrogen-deuterium exchange mass spectrometry) to identify substrate interaction regions

Based on studies of the E. coli methionine ABC transporter, researchers should particularly investigate the transport of D-methionine derivatives, as the MetNI system has shown broad specificity toward methionine derivatives, including D-methionine . Additionally, the noncanonical role of binding proteins observed in E. coli MetNI transport of D-selenomethionine suggests examining similar phenomena in the P. profundum system .

How can mutagenesis approaches be used to study structure-function relationships in P. profundum MetN?

Systematic mutagenesis approaches provide powerful tools for elucidating structure-function relationships in P. profundum MetN:

• Targeted Site-Directed Mutagenesis:

  • Walker A motif (e.g., K45A) to disrupt ATP binding

  • Walker B motif (e.g., D170N) to allow ATP binding but prevent hydrolysis

  • ABC signature motif mutations to disrupt dimer formation

  • C-terminal regulatory domain mutations to alter trans-inhibition

• Alanine-Scanning Mutagenesis:

  • Systematic replacement of residues with alanine along potential substrate paths

  • Focus on residues at the NBD-TMD interface that may be involved in conformational coupling

• Domain Swapping Experiments:

  • Replace domains with homologous regions from pressure-sensitive organisms (e.g., E. coli MetN)

  • Create chimeric proteins to identify pressure-adaptation determinants

  • Exchange regulatory domains to investigate specificity of trans-inhibition

• Functional Analysis of Mutants:

  • ATPase activity assays under varying pressure conditions

  • Transport assays using reconstituted systems

  • Thermal and pressure stability measurements

  • Binding affinity determination for ATP and substrate

• In vivo Complementation Studies:

  • Develop genetic systems in P. profundum similar to those used for flagellar gene studies

  • Create metN deletion strains using suicide vectors like pRL271

  • Test mutant constructs for complementation under varying pressure conditions

This approach should be guided by the successful genetic manipulation strategies previously employed in P. profundum, where in-frame deletions have been constructed using a two-step recombination process with the sacB-containing suicide vector pRL271 .

How does P. profundum MetN compare structurally and functionally with its E. coli homolog?

A comparative analysis of P. profundum MetN and E. coli MetN reveals both conserved features and adaptations specific to each organism's ecological niche:

• Sequence and Structural Comparison:

  • Core NBD domains likely show high conservation in ATP-binding motifs

  • The C-terminal regulatory domain may exhibit differences reflecting distinct regulatory mechanisms

  • Pressure-adapted residues in P. profundum MetN may include increased hydrophobic packing and reduced void volumes

• Functional Differences:

  • E. coli MetNI exhibits high-affinity transport toward L-methionine and broad specificity toward methionine derivatives

  • P. profundum MetN likely shows pressure-optimized kinetic parameters, potentially with higher catalytic efficiency at elevated pressures

  • The substrate specificity profile may differ, reflecting adaptation to deep-sea nutrient conditions

• Regulatory Mechanisms:

  • While E. coli MetNI is known to be regulated by trans-inhibition, where intracellular methionine binds to the C-terminal domain and inhibits transport

  • P. profundum MetN might have evolved modified regulatory mechanisms optimized for deep-sea conditions and pressure fluctuations

• Interaction with Binding Proteins:

  • E. coli studies have revealed a noncanonical role for the binding protein in substrate uptake, particularly for D-selenomethionine transport

  • The interaction between P. profundum MetN and its cognate binding protein (MetQ) may show distinct characteristics reflecting evolutionary adaptations

The noncanonical role of binding proteins observed in E. coli methionine transport provides an interesting avenue for investigation in P. profundum, potentially revealing convergent or divergent evolutionary solutions to similar functional challenges .

What unique adaptations might P. profundum MetN exhibit for functioning in high-pressure environments?

Based on knowledge of deep-sea adaptations in P. profundum and other piezophiles, MetN likely exhibits several pressure-adaptive features:

P. profundum SS9 has demonstrated remarkable pressure adaptation in its motility systems, maintaining function up to 150 MPa while pressure-sensitive bacteria like E. coli show dramatic decreases in functionality . Similar adaptations are likely present in the methionine transport system, potentially allowing it to function optimally at the organism's preferred pressure of approximately 28 MPa.

How do the genetics and regulation of metN expression differ between pressure-adapted and pressure-sensitive bacterial species?

The regulation and expression patterns of metN likely exhibit important differences between pressure-adapted P. profundum and pressure-sensitive bacteria:

• Transcriptional Regulation:

  • Pressure-adapted species may have evolved pressure-responsive promoter elements

  • RNA polymerase sigma factors like those identified in P. profundum 3TCK (P3TCK_10673; RNA polymerase sigma factor, ECF subfamily) may play roles in pressure-responsive transcription

  • Pressure-adapted bacteria might employ specialized transcription factors that sense pressure changes

• Expression Patterns:

  • P. profundum likely shows altered metN expression patterns under different pressure conditions

  • Similar to the lateral flagellum system in P. profundum SS9, which is induced by high pressure and viscosity , metN may show pressure-dependent expression

  • Expression might be coordinated with other transport systems and metabolic pathways affected by pressure

• Genetic Context:

  • The genomic organization around metN may differ between pressure-adapted and pressure-sensitive species

  • Operon structures and genetic linkage to other pressure-responsive genes might be observed

  • Horizontal gene transfer, which has been suggested for some P. profundum genes (like the lateral flagellum cluster with higher GC content) , might also have influenced metN evolution

• Post-transcriptional Regulation:

  • RNA secondary structures may differ to maintain stability under pressure

  • Translation efficiency might be optimized for high-pressure conditions

  • Post-translational modifications could play roles in pressure adaptation

Experimental approaches similar to those used for studying flagellar gene expression in P. profundum SS9, where pressure and viscosity-induced expression of flaB and motA1 genes was observed , could be adapted to investigate metN regulation under varying pressure conditions.

How can P. profundum MetN be utilized in structural studies of pressure adaptation mechanisms?

P. profundum MetN represents an excellent model system for structural studies of pressure adaptation mechanisms:

• High-Resolution Structural Analysis:

  • X-ray crystallography at different pressures using pressure cells

  • Cryo-EM studies of the complete transporter complex under native-like conditions

  • NMR spectroscopy to analyze protein dynamics under varying pressure

  • HDX-MS (hydrogen-deuterium exchange mass spectrometry) to map pressure-sensitive regions

• Comparative Structural Biology:

  • Parallel structural studies of MetN from pressure-adapted and pressure-sensitive organisms

  • Analysis of structural differences in ATP-binding pockets, dimer interfaces, and regulatory domains

  • Identification of specific amino acid substitutions responsible for pressure adaptation

• Structure-Based Computational Approaches:

  • Molecular dynamics simulations under varying pressure conditions

  • Free energy calculations to determine energetic contributions to pressure stability

  • Evolutionary analysis to identify positively selected residues in piezophiles

• Methodological Development:

  • Establishing protocols for membrane protein crystallization under pressure

  • Developing specialized equipment for structural biology under pressure

  • Creating reporter systems for conformational changes under pressure

The crystal structure of the E. coli MetNI transporter at 2.95 Å resolution provides a valuable template for comparative structural studies, enabling identification of specific adaptations in the P. profundum homolog.

What experimental controls are essential when working with recombinant P. profundum MetN?

Rigorous experimental controls are critical for research involving recombinant P. profundum MetN:

• Negative Controls:

  • Inactive mutants (e.g., Walker A/B mutations) to verify specific enzymatic activity

  • Empty vector controls for expression studies

  • Heat-denatured protein for binding and activity assays

  • Inhibitor-treated samples (e.g., vanadate for ATPase inhibition)

• Positive Controls:

  • Well-characterized ABC transporters (e.g., E. coli MetN) tested under identical conditions

  • Commercial ATPases for standard curve generation in activity assays

  • Previously validated protein preparations as batch-to-batch controls

• System-Specific Controls:

  • Atmospheric pressure controls for all high-pressure experiments

  • Pressure-sensitive protein controls (e.g., E. coli proteins) in parallel with P. profundum MetN

  • Thermal stability controls at different pressures to distinguish pressure effects from temperature effects

• Methodological Controls:

  • Multiple buffer conditions to ensure results aren't buffer-specific

  • Varied protein concentrations to identify concentration-dependent artifacts

  • Different expression systems to verify native folding and function

  • Storage condition controls (fresh vs. frozen protein)

• Validation Approaches:

  • Orthogonal methods for key measurements

  • Multiple substrate analogs to confirm specificity patterns

  • Replication across multiple protein preparations

  • In vivo validation of key findings using genetic approaches

A systematic control strategy should be documented in a form similar to this validation matrix:

How can researchers design experiments to elucidate the interaction between P. profundum MetN and other components of the methionine transport system?

Investigating interactions between MetN and other components of the methionine transport system requires a multi-faceted experimental design:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation of tagged components from native membranes

  • Bacterial two-hybrid assays for interaction mapping

  • FRET-based interaction studies in reconstituted systems

  • Cross-linking mass spectrometry to identify interaction interfaces

  • Surface plasmon resonance (SPR) for binding kinetics between purified components

• Functional Coupling Analysis:

  • ATPase stimulation assays in the presence of MetI and MetQ components

  • Transport assays in proteoliposomes with systematically varied component ratios

  • Thermodynamic coupling measurements using ITC

  • Pressure-dependent alterations in component interactions

• Structural Approaches:

  • Cryo-EM of the complete transporter complex

  • Cross-linking followed by structural analysis

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Comparative structural analysis of isolated components versus complexes

• Genetic Approaches:

  • Construction of P. profundum strains with mutations in different transporter components

  • Synthetic lethality screening to identify genetic interactions

  • Suppressor mutation analysis to identify compensatory adaptations

  • Similar to the approach used for studying fabD and pfaA synthetic lethality in P. profundum SS9

• Chimeric Protein Analysis:

  • Domain swapping between P. profundum and E. coli components

  • Creation of hybrid transporters to identify critical interaction determinants

  • Heterologous expression systems to test cross-species component compatibility

  • Analogous to experiments demonstrating that heterologous expression of P. profundum SS9 Pfa synthase could complement E. coli fabD mutations

The experimental design should be informed by successful genetic manipulation strategies previously employed in P. profundum, including the use of suicide vectors like pRL271 for gene deletion and complementation studies .

What are common pitfalls in recombinant expression of P. profundum MetN and how can they be overcome?

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

• Low Expression Yields:

  • Problem: Deep-sea proteins often express poorly in standard systems

  • Solution: Optimize codons for expression host; use specialized strains like ArcticExpress; reduce expression temperature to 15-20°C; try autoinduction media; consider high-pressure expression systems for proper folding

• Protein Insolubility:

  • Problem: Formation of inclusion bodies

  • Solution: Express as fusion protein with solubility tags (MBP, SUMO); co-express with chaperones; use mild detergents (0.05% DDM or LMNG) during lysis; try on-column refolding approaches

• Loss of Activity During Purification:

  • Problem: Protein loses ATPase activity during purification

  • Solution: Include stabilizing agents (5-10% glycerol, 1-2 mM ATP); reduce purification time; maintain low temperature throughout; avoid freeze-thaw cycles; consider adding lipids to stabilize membrane-associated regions

• Pressure-Related Structural Changes:

  • Problem: Protein may adopt non-native conformations at atmospheric pressure

  • Solution: Perform key characterization steps under moderate pressure (10-30 MPa); validate findings using pressure-stable and pressure-sensitive controls; consider rapid analysis immediately after decompression

• Aggregation During Storage:

  • Problem: Protein aggregates during storage

  • Solution: Add stabilizing agents; store at higher concentrations (>1 mg/ml); avoid freezing if possible; if freezing is necessary, flash-freeze in liquid nitrogen with cryoprotectants; aliquot to avoid repeated freeze-thaw cycles

The table below summarizes troubleshooting strategies for common issues:

How can researchers validate that recombinant P. profundum MetN retains native-like structure and function?

Comprehensive validation of recombinant P. profundum MetN requires multiple complementary approaches:

• Structural Validation:

  • Circular dichroism (CD) spectroscopy to verify secondary structure content

  • Thermal denaturation profiles to assess stability

  • Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to verify oligomeric state

  • Limited proteolysis patterns to assess domain organization

  • Intrinsic fluorescence spectroscopy to examine tertiary structure

• Functional Validation:

  • ATPase activity measurements under various conditions

  • Nucleotide binding assays (ITC, fluorescence-based)

  • Transport assays in reconstituted systems

  • Response to known modulators (substrate, inhibitors)

  • Pressure-dependent activity profiles

• Comparative Validation:

  • Direct comparison with native protein (if available)

  • Comparison with homologous proteins under identical conditions

  • Verification that activity patterns correlate with physiological conditions (e.g., optimal activity at 28-30 MPa pressure)

  • Similar methodological approach to that used for validating P. profundum SS9 motility at different pressures

• In vivo Validation:

  • Complementation of metN knockout mutants

  • Phenotypic rescue under varying pressure conditions

  • In vivo transport assays using radioactive substrates

  • Similar approach to the methodology used for validating flagellar gene function in P. profundum

• Specific Controls for Pressure Adaptation:

  • Comparative pressure stability versus pressure-sensitive homologs

  • Pressure-activity profiles matching organismal growth optima

  • Reversibility of pressure effects on structure and function

A systematic validation approach should demonstrate that the recombinant protein exhibits the expected pressure-optimized characteristics observed in other P. profundum proteins, such as maintained function at pressures up to 150 MPa as seen with the flagellar motility system .

What specialized equipment is needed for studying pressure effects on recombinant P. profundum MetN?

Research on pressure effects requires specialized equipment and technical considerations:

• High-Pressure Enzymatic Assay Systems:

  • Pressure vessels with optical windows for spectrophotometric assays

  • Temperature-controlled high-pressure chambers

  • Rapid pressure cycling systems for kinetic studies

  • Online monitoring capabilities for real-time measurements

  • Similar to the HPDS high-pressure cell system used for motility studies

• High-Pressure Structural Biology Equipment:

  • High-pressure crystallography setup

  • Pressure-adapted NMR tubes and equipment

  • Diamond anvil cells for spectroscopic studies

  • Pressure-compatible sample holders for cryo-EM

• Specialized Biochemical Equipment:

  • High-pressure stopped-flow apparatus

  • Pressure-resistant fluorescence cuvettes

  • Systems for sample extraction under pressure

  • Equipment for maintaining pressure during protein purification

• Safety and Control Systems:

  • Pressure monitoring and control systems

  • Safety shields and containment systems

  • Automated pressure relief mechanisms

  • Calibration standards for pressure measurements

• Additional Specialized Resources:

  • High-pressure growth chambers for expressing protein under native-like conditions

  • Pressure-cycling incubators for adaptation studies

  • Computational resources for molecular dynamics under pressure

  • Equipment for measuring physical properties (volume, compressibility) under pressure

An example setup for high-pressure enzymatic studies would include:

• Stainless steel pressure vessel with sapphire windows

• Fiber optic spectrophotometer probe

• Temperature control system (±0.1°C)

• Pressure generation system (hydraulic pump or gas compressor)

• Pressure transducer and data acquisition system

• Sample cell with mixing capability

• Safety pressure relief valve

This specialized equipment represents a significant investment but is essential for accurately characterizing the pressure-adapted properties of P. profundum MetN and comparing them with pressure-sensitive homologs.