Recombinant Photobacterium profundum Bifunctional protein FolD (folD)

What is the Photobacterium profundum bifunctional protein FolD and what are its primary functions?

The bifunctional protein FolD from Photobacterium profundum is an enzyme involved in folate metabolism that catalyzes two sequential reactions in the one-carbon metabolic pathway. It possesses both methylenetetrahydrofolate dehydrogenase (MTHFD) and methenyltetrahydrofolate cyclohydrolase (MTHFC) activities. The enzyme plays a critical role in cellular one-carbon metabolism, which is essential for nucleotide biosynthesis, amino acid metabolism, and methylation reactions .

The protein is of particular interest due to P. profundum's status as a model piezophilic organism that thrives under high-pressure conditions (optimal growth at 28 MPa and 15°C) . This environmental adaptation suggests that its FolD protein may possess unique structural and functional properties compared to homologous proteins from mesophilic organisms.

How does P. profundum FolD differ from homologous proteins in non-piezophilic organisms?

P. profundum FolD likely exhibits structural adaptations that enable functionality under high-pressure conditions. While specific details about P. profundum FolD are not extensively documented, research on other proteins from this organism has revealed pressure-adaptive features that may also apply to FolD:

• Increased structural flexibility in key regions to maintain catalytic activity under pressure

• Modified amino acid composition with fewer salt bridges and increased hydrophobic interactions

• Potential alterations in substrate binding sites to accommodate pressure-induced conformational changes

Proteomic studies of P. profundum have demonstrated differential expression of various metabolic enzymes under high versus atmospheric pressure conditions, suggesting that folate metabolism enzymes like FolD may be regulated in response to pressure changes .

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

The expression of recombinant P. profundum FolD presents unique challenges due to its origin from a piezophilic organism. Based on successful approaches with other deep-sea proteins, the following expression systems are recommended:

For optimal results, expression in E. coli strains should be conducted at lower temperatures (15-18°C) with reduced inducer concentrations to allow proper folding. The thermostable exoshell system has been shown to improve expression and stabilization of recombinant proteins through steric accommodation and charge complementation, potentially increasing functional folding yields by ~100-fold .

What purification strategies work best for obtaining active P. profundum FolD?

Purification of active P. profundum FolD requires protocols that maintain the protein's native conformation throughout the process:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using His-tagged constructs under mild conditions (pH 7.5-8.0, 300-400 mM NaCl)

• Intermediate purification: Ion exchange chromatography at 4°C with gradual salt gradient elution

• Polishing step: Size exclusion chromatography to obtain homogeneous protein preparation

• Buffer optimization: Include osmolytes (glycerol 5-10%, TMAO 1-2 M) that mimic high-pressure effects

Throughout purification, maintaining lower temperatures (4-10°C) and including pressure-mimicking osmolytes can help preserve the native state of the protein. For activity assays, consider performing the experiments under various pressure conditions using specialized high-pressure chambers similar to those used in P. profundum motility studies .

How does hydrostatic pressure affect the structure and function of P. profundum FolD?

Hydrostatic pressure likely influences both the structure and catalytic properties of P. profundum FolD in several ways:

• Conformational stability: Similar to other P. profundum proteins, FolD likely maintains structural integrity under pressure conditions that would denature mesophilic homologs. This adaptation may involve specific amino acid substitutions that favor compact protein packing.

• Catalytic efficiency: Kinetic parameters (Km, kcat) of P. profundum FolD may show optimal values under elevated pressure. Pressure-dependent activity profiles should exhibit a bell-shaped curve with maximal activity near 28 MPa, corresponding to the organism's native environment.

• Substrate binding: Pressure may alter substrate binding pocket geometry, potentially necessitating adaptations in residues involved in substrate recognition.

Research approaches to investigate these effects should include activity assays performed in high-pressure chambers, structural analysis using pressure-resistant spectroscopic methods (fluorescence, CD spectroscopy), and comparative molecular dynamics simulations under varying pressure conditions .

What techniques are appropriate for studying the thermal and pressure stability of P. profundum FolD?

Given P. profundum's adaptation to both cold temperatures and high pressure, multiple complementary techniques should be employed to characterize FolD stability:

Particularly informative would be the comparison of stability parameters between atmospheric (0.1 MPa) and optimal growth pressure (28 MPa) conditions. Based on studies of other P. profundum proteins, FolD likely exhibits enhanced stability at higher pressures, with potential cold adaptation features allowing function at low temperatures .

How has the folD gene evolved in P. profundum to function optimally under high-pressure conditions?

Evolutionary adaptations in P. profundum folD likely include:

• Amino acid substitutions: Comparative genomic analyses of folD genes across pressure-adapted and non-adapted bacteria would reveal pressure-specific substitutions, particularly in flexible regions and at the active site.

• Codon usage optimization: P. profundum may employ specific codon biases for efficient translation under high-pressure conditions, especially for highly expressed genes like folD.

• Regulatory elements: The promoter and regulatory regions of folD may contain pressure-responsive elements similar to those identified in other pressure-regulated genes in P. profundum.

Transposon mutagenesis studies of P. profundum have demonstrated that many genes show differential expression under varying pressure conditions . While folD was not specifically identified in these studies, similar regulatory mechanisms may apply to this gene. Phylogenetic analysis comparing folD sequences from shallow-water and deep-sea Vibrionaceae members would provide insights into the evolutionary trajectory of pressure adaptation .

Does P. profundum contain multiple isoforms of folD adapted to different pressure conditions?

While the search results don't specifically address folD isoforms in P. profundum, the organism's ability to grow across a wide pressure range (0.1-90 MPa) suggests possible pressure-specific protein variants. Studies on P. profundum's flagellar systems provide a relevant parallel:

P. profundum possesses two distinct flagellar systems, both of which have acquired adaptations for optimal functionality under high-pressure conditions . Similar gene duplication and specialization may have occurred for metabolic genes like folD. Proteomic analysis has shown differential protein expression between atmospheric and high-pressure growth conditions .

To investigate potential folD isoforms:

• Conduct genomic analysis to identify potential gene duplications

• Perform RNA-seq under varying pressure conditions to detect differential expression

• Use proteomic approaches to identify pressure-specific protein variants

• Express and characterize any identified isoforms to determine their pressure-activity relationships

What specialized equipment is required for studying P. profundum FolD under native pressure conditions?

Research on P. profundum FolD under high-pressure conditions requires specialized equipment:

• High-pressure cultivation systems: Custom-designed stainless steel pressure vessels (such as those described for P. profundum mutagenesis screening) capable of maintaining 28-45 MPa pressure for extended periods .

• High-pressure microscopic chambers: Specialized chambers for direct observation of protein behavior under pressure, similar to those used for swimming velocity measurements of P. profundum .

• Pressure-resistant spectroscopic cells: Equipped with sapphire or diamond windows for spectroscopic measurements (fluorescence, absorbance) under pressure.

• Pressure perturbation calorimetry: For thermodynamic analysis of pressure effects on protein stability and function.

• High-pressure stopped-flow apparatus: For kinetic measurements of enzymatic activity under varying pressure conditions.

When designing experiments, consider that macromolecular crowding significantly impacts protein folding and function in cellular environments . In vitro systems should attempt to mimic not only the pressure conditions but also the crowded cellular environment of P. profundum.

How can researchers differentiate between direct pressure effects on FolD and indirect effects mediated by the cellular environment?

Distinguishing direct versus indirect pressure effects requires a multi-layered experimental approach:

• Purified protein studies: Characterize isolated recombinant FolD under varying pressure conditions to identify intrinsic pressure responses.

• Reconstituted systems: Incorporate purified FolD into artificial cellular environments with controlled composition (lipids, osmolytes, macromolecular crowding agents) to identify interaction-dependent effects.

• In-cell studies: Use techniques like split-reporter assays (similar to those developed for other proteins) to monitor FolD folding and activity in living cells under pressure .

• Comparative approaches: Study FolD variants from related non-piezophilic organisms under identical conditions to identify pressure-specific adaptations.

• Site-directed mutagenesis: Systematically modify pressure-adaptive residues to quantify their contribution to pressure resistance.

In interpreting results, consider that macromolecular crowding alters protein folding pathways and can shift denatured and intermediate ensembles away from extended states . Effects observed under high pressure may reflect complex interactions between direct pressure effects, changes in cellular crowding, and alterations in molecular interactions.

How can the pressure-adaptive features of P. profundum FolD be applied to enhance the stability of proteins for biotechnological applications?

The pressure-adaptive features of P. profundum FolD represent valuable design principles for protein engineering:

• Engineered enzymes: Transferring identified pressure-adaptive residues to mesophilic homologs could create enzymes with enhanced stability for industrial biocatalysis.

• Pharmaceutical proteins: Incorporating pressure-adaptive features could improve the storage stability and shelf-life of therapeutic proteins.

• Biosensors: Development of pressure-resistant biosensors based on FolD structural motifs for deep-sea and high-pressure industrial monitoring.

• Thermostable exoshell technology: The principles used to create thermostable protein nanoparticles that improve expression and stabilization of recombinant proteins could be enhanced with pressure-adaptive features from P. profundum FolD .

Specific applications might include using P. profundum FolD as a model for designing enzymes that retain activity under extreme conditions or incorporating its pressure-adaptive features into protein therapeutics to enhance stability during manufacturing and storage.

What fundamental insights about protein adaptation can be gained by comparing P. profundum FolD with homologs from non-piezophilic organisms?

Comparative analysis of P. profundum FolD with homologs from non-piezophilic organisms can provide insights into:

• Molecular basis of pressure adaptation: Identifying specific residues and structural elements that confer pressure resistance.

• Evolutionary pathways: Understanding how proteins evolve under selective pressure from extreme environments.

• Structure-function relationships: Determining how structural adaptations maintain enzymatic function under pressure.

• Protein energy landscapes: Elucidating how pressure affects the conformational energy landscape of proteins.

• Cellular homeostasis: Understanding how organisms maintain metabolic balance under extreme conditions.

Research approaches should incorporate structural biology (X-ray crystallography, cryo-EM), molecular dynamics simulations, and functional assays under varying pressure conditions. The findings would contribute to our fundamental understanding of protein biophysics and potentially reveal general principles of adaptation to extreme environments that could inform synthetic biology and protein design efforts.

What are the most common challenges in expressing and purifying active recombinant P. profundum FolD and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant P. profundum FolD:

For particularly difficult cases, consider using the thermostable exoshell (tES) approach, which has been shown to improve both expression and stabilization of recombinant proteins through steric accommodation and charge complementation . This technology could be especially relevant for a pressure-adapted enzyme like P. profundum FolD.

How can researchers validate that recombinant P. profundum FolD retains its native structure and function after expression and purification?

Validation of recombinant P. profundum FolD structural and functional integrity requires multiple complementary approaches:

• Enzymatic activity assays: Compare specific activity and kinetic parameters (Km, kcat) at various pressures (0.1-45 MPa) to determine if activity profiles match expected pressure dependence.

• Circular dichroism (CD) spectroscopy: Verify secondary structure content matches theoretical predictions based on sequence.

• Thermal shift assays: Compare melting temperatures at atmospheric pressure and elevated pressures (requires specialized equipment).

• Size exclusion chromatography: Confirm proper oligomeric state and absence of aggregation.

• Mass spectrometry: Verify intact mass and post-translational modifications.

• Limited proteolysis: Compare digestion patterns of recombinant protein with native protein (if available).

• Substrate binding studies: Determine if substrate binding affinity shows expected pressure dependence.

Compare these parameters with data from related FolD proteins while accounting for expected differences due to pressure adaptation. Consider that P. profundum is under greater stress at atmospheric pressure than at elevated pressure, reflecting its deep-sea origin , which may affect protein behavior during standard laboratory manipulations.