Recombinant Photobacterium profundum UPF0283 membrane protein PBPRA2435 (PBPRA2435)

How does PBPRA2435 relate to other membrane proteins in Photobacterium profundum?

PBPRA2435 belongs to the UPF0283 family of membrane proteins found in various bacterial species. In Photobacterium profundum, this protein may play a role in the organism's adaptation to high-pressure environments, as P. profundum is a piezophilic (pressure-loving) bacterium . Gene expression studies of P. profundum have shown that certain membrane proteins, potentially including PBPRA2435, are differentially expressed under varying pressure conditions.

Research on P. profundum has identified different ecotypes with varying genetic adaptations. These ecotypes show distinct phenotypic characteristics, including differences in membrane composition and protein expression profiles that may affect the expression and function of membrane proteins like PBPRA2435 . Understanding these relationships requires consideration of the organism's environmental adaptations and the functional role of its membrane proteins in response to pressure, temperature, and other environmental factors.

What prediction methods are most reliable for determining PBPRA2435 membrane topology?

For membrane proteins like PBPRA2435, multiple computational and experimental approaches should be combined to predict topology with confidence:

For PBPRA2435 specifically, combining transmembrane prediction algorithms with homology modeling based on related UPF0283 family proteins would provide the most reliable topology prediction. Follow-up validation using biochemical approaches is essential to confirm these predictions, particularly regarding the orientation of the N- and C-termini and the number of membrane-spanning segments .

What expression systems yield optimal results for recombinant PBPRA2435?

E. coli remains the primary expression system for PBPRA2435, with successful expression reported using N-terminal His-tagged constructs . The expression protocol typically involves:

• Transformation of expression vector into an appropriate E. coli strain (BL21(DE3), C41(DE3), or C43(DE3))

• Culture in LB or 2xYT medium supplemented with appropriate antibiotics

• Induction with IPTG (0.1-1.0 mM) at optical density (OD600) of 0.6-0.8

• Post-induction growth at reduced temperature (16-25°C) for 4-18 hours

• Cell harvesting by centrifugation and storage at -80°C until purification

For membrane proteins like PBPRA2435, expression levels can be improved by:

• Using specialized E. coli strains designed for membrane protein expression

• Optimizing induction conditions (temperature, IPTG concentration, duration)

• Including membrane-stabilizing agents in the growth medium

• Employing auto-induction media for gradual protein expression

When designing expression experiments, researchers should carefully consider parameters such as expression strain, induction conditions, and harvest timing to optimize protein yield and quality . While E. coli is commonly used, alternative expression systems like Pichia pastoris might be considered for cases where proper folding is challenging in bacterial systems.

What purification strategies yield the highest purity and activity of recombinant PBPRA2435?

The purification of PBPRA2435 requires specialized approaches for membrane proteins:

• Membrane isolation and solubilization:

  • Cell lysis via sonication, French press, or enzymatic methods

  • Membrane fraction isolation by ultracentrifugation (100,000 × g, 1 hour)

  • Solubilization with detergents (typically 1-2% n-dodecyl-β-D-maltoside (DDM), LDAO, or Triton X-100)

  • Incubation with gentle agitation for 1-2 hours at 4°C

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Equilibration buffer containing 20-50 mM imidazole and 0.05-0.1% detergent

  • Wash steps with increasing imidazole (50-100 mM)

  • Elution with 250-500 mM imidazole

• Secondary purification:

  • Size exclusion chromatography to remove aggregates and achieve higher purity

  • Ion exchange chromatography as an additional purification step

• Quality assessment:

  • SDS-PAGE analysis for purity (>90% as reported for commercial preparations)

  • Western blot for identity confirmation

  • Circular dichroism for secondary structure verification

For optimal results, researchers should maintain the protein in detergent-containing buffers throughout the purification process to prevent aggregation . The choice of detergent is critical and may require optimization for PBPRA2435 specifically.

How can researchers properly store and handle purified PBPRA2435 to maintain stability?

Proper storage and handling of purified PBPRA2435 is crucial for maintaining protein stability and function:

• Short-term storage (up to one week):

  • Store at 4°C in purification buffer containing detergent

  • Avoid repeated freeze-thaw cycles which can lead to protein denaturation

• Long-term storage:

  • Aliquot to avoid repeated freeze-thaw cycles

  • Store at -20°C or preferably -80°C

  • Include cryoprotectants such as glycerol (final concentration 5-50%)

  • Lyophilization is an option but requires optimization of buffer components

• Reconstitution from lyophilized form:

  • Briefly centrifuge vial before opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol (final concentration 5-50%) for aliquoting and storage

• Quality control during storage:

  • Monitor protein integrity by SDS-PAGE before use

  • Assess functional activity using appropriate assays

  • Check detergent concentration, as detergent depletion can cause aggregation

The storage buffer composition significantly impacts stability. Tris/PBS-based buffer with 6% trehalose at pH 8.0 has been reported as effective for PBPRA2435 . Researchers should validate storage conditions for their specific experimental requirements, particularly if functional assays will be performed.

What critical factors should be considered when designing experiments for PBPRA2435 functional analysis?

Designing robust experiments for PBPRA2435 functional analysis requires careful consideration of multiple factors:

• Experimental variables control:

  • Independent variables: protein concentration, substrate concentration, temperature, pH, detergent type/concentration

  • Dependent variables: activity measurements, binding affinities, structural parameters

  • Control variables: buffer composition, salt concentration, presence of stabilizing agents

• Experimental controls:

  • Positive controls: well-characterized membrane proteins with known function

  • Negative controls: denatured protein, empty liposomes, non-functional mutants

  • Vehicle controls: detergent-only samples to account for detergent effects

• Statistical considerations:

  • Sample size determination through power analysis

  • Randomization and blinding procedures where applicable

  • Appropriate statistical tests for data analysis

• Membrane environment:

  • Detergent micelles vs. proteoliposomes vs. nanodiscs

  • Lipid composition effects on protein activity

  • Protein orientation in reconstituted systems

When designing experiments, researchers should apply design of experiments (DOE) principles to systematically explore the experimental space and identify key parameters affecting PBPRA2435 function . This approach allows for efficient optimization of conditions while minimizing the number of experiments required.

How should researchers approach reconstitution of PBPRA2435 into proteoliposomes?

Reconstitution of PBPRA2435 into proteoliposomes requires careful attention to methodology:

• Preparation of liposomes:

  • Select appropriate lipid composition (consider native membrane composition of P. profundum)

  • Prepare liposomes by film hydration, extrusion, or reverse-phase evaporation

  • Size liposomes by extrusion through polycarbonate filters (typically 100-400 nm)

• Reconstitution methods:

  • Detergent-mediated reconstitution: gradually remove detergent using Bio-Beads, dialysis, or gel filtration

  • Direct incorporation during liposome formation

  • Protein:lipid ratio optimization (typically 1:50 to 1:1000 w/w)

• Control of protein orientation:

  • Protein orientation in liposomes is typically random and difficult to predict

  • Methods to influence orientation include:

    • Use of fusion domains (as demonstrated with other membrane proteins)

    • Immobilization on Ni-NTA beads prior to reconstitution

    • Asymmetric reconstitution using pH or ionic gradients

• Quality assessment:

  • Freeze-fracture electron microscopy to visualize protein incorporation

  • Dynamic light scattering for size distribution

  • Density gradient centrifugation to separate proteoliposomes from free protein

  • Functional assays to verify activity after reconstitution

The orientation of PBPRA2435 in proteoliposomes is a critical consideration, especially if the protein has directional functionality. Research indicates that orientation can be influenced by reconstitution method, lipid composition, and protein characteristics, though the outcome remains difficult to predict for novel proteins .

What analytical methods are most suitable for studying protein-lipid interactions involving PBPRA2435?

Investigating PBPRA2435-lipid interactions requires specialized analytical approaches:

When studying PBPRA2435-lipid interactions, researchers should consider both bulk membrane effects and specific binding of individual lipid molecules. The choice of analytical method depends on the specific research question, available equipment, and sample quantities. Multiple complementary approaches are recommended for comprehensive characterization .

What assays can be used to determine the functional activity of PBPRA2435?

While the specific function of PBPRA2435 is not fully characterized, several approaches can be used to investigate its activity:

• Transport assays (if PBPRA2435 functions as a transporter):

  • Fluorescence-based assays using pH-sensitive or ion-sensitive dyes

  • Radioactive substrate uptake measurements

  • Liposome swelling assays to detect osmolyte transport

• Binding assays:

  • Surface plasmon resonance (SPR) to detect ligand binding

  • Isothermal titration calorimetry (ITC) for binding thermodynamics

  • Microscale thermophoresis for binding in detergent solutions

• Structural changes upon activation:

  • Fluorescence resonance energy transfer (FRET) to detect conformational changes

  • Electron paramagnetic resonance (EPR) with site-directed spin labeling

  • Limited proteolysis to identify exposed regions in different functional states

• Phenotypic assays:

  • Complementation of knockout strains

  • Growth assays under various stress conditions

  • Pressure adaptation studies in P. profundum

For membrane proteins with unknown function like PBPRA2435, a combination of candidate-based approaches (testing predicted substrates) and unbiased screening methods may be necessary to determine function. Researchers should consider the native environment of P. profundum (high pressure, marine conditions) when designing functional assays.

How can researchers effectively study protein-protein interactions involving PBPRA2435?

Investigating protein-protein interactions involving membrane proteins like PBPRA2435 requires specialized approaches:

• In vitro methods:

  • Pull-down assays using His-tagged PBPRA2435 as bait

  • Co-immunoprecipitation with antibodies against PBPRA2435 or potential partners

  • Crosslinking followed by mass spectrometry (XL-MS)

  • Förster resonance energy transfer (FRET) between labeled proteins

• Reconstitution-based approaches:

  • Co-reconstitution with potential partner proteins in proteoliposomes

  • Functional coupling assays to detect cooperative activity

  • Freeze-fracture electron microscopy to visualize protein complexes

• In vivo methods:

  • Bacterial two-hybrid systems adapted for membrane proteins

  • Fluorescence-based protein complementation assays

  • Co-expression followed by tandem affinity purification

• Computational predictions:

  • Structural modeling of potential interaction interfaces

  • Sequence covariation analysis to identify co-evolving residues

  • Protein-protein docking simulations

When co-reconstituting PBPRA2435 with other proteins, researchers must consider the orientation of both proteins in the membrane, as this significantly affects their ability to interact . Tagged versions of the proteins can be used to verify incorporation and correct orientation in proteoliposomes.

What approaches can be used to determine the effect of pressure on PBPRA2435 structure and function?

Given that P. profundum is a piezophilic organism, studying PBPRA2435 under pressure is particularly relevant:

When designing pressure experiments, researchers should consider both the physiological pressure range of P. profundum (found at depths up to 5000 meters) and experimental capabilities. Controls should include well-characterized pressure-sensitive and pressure-insensitive proteins to benchmark responses .

How can de novo design principles be applied to study or modify PBPRA2435?

Recent advances in de novo membrane protein design offer powerful approaches for studying PBPRA2435:

• Structure-based redesign:

  • Computational redesign of transmembrane segments for improved stability

  • Introduction of novel binding or catalytic sites

  • Simplification of complex regions to create minimal functional units

• Function-based modifications:

  • Design of chimeric proteins combining PBPRA2435 with functional domains from other proteins

  • Creation of biosensors by introducing environment-sensitive fluorophores

  • Engineering of dimerization interfaces to control protein assembly

• Applying recent design advances:

  • Implementation of multipass transmembrane protein design principles

  • Application of "rocket-shaped" structure designs with wide cytoplasmic base funneling into transmembrane helices

  • Utilization of de novo designed stable scaffold proteins

As demonstrated in recent research, computational design now allows creation of complex transmembrane proteins with multiple membrane-spanning regions that form specific oligomeric assemblies . These principles can be applied to PBPRA2435 to create variants with altered topology, oligomerization state, or function, providing valuable insights into structure-function relationships.

What strategies are effective for studying PBPRA2435 in cellular contexts?

Investigating PBPRA2435 in cellular contexts requires specific approaches:

• Heterologous expression systems:

  • Expression in E. coli with verification of membrane localization

  • Adaptation to expression in eukaryotic cells to study trafficking

  • Use of inducible expression systems to control protein levels

• Cellular localization studies:

  • Fluorescent protein fusions to track localization

  • Immunofluorescence with antibodies against PBPRA2435 or epitope tags

  • Subcellular fractionation followed by Western blotting

• Functional analysis in cells:

  • Complementation of knockout strains

  • Phenotypic assays under various stress conditions

  • Metabolomic analysis to detect changes in cellular metabolites

• Protein-protein interactions in cellular context:

  • Proximity-based labeling methods (BioID, APEX)

  • Förster resonance energy transfer (FRET) in living cells

  • Co-immunoprecipitation from membrane fractions

Recent developments in de novo designed membrane proteins have demonstrated successful localization to the plasma membrane in both bacterial and mammalian cells . Similar approaches can be applied to PBPRA2435 to study its trafficking, localization, and function in different cellular environments.

How can researchers investigate the relationship between PBPRA2435 and bacterial adaptation to extreme environments?

P. profundum is adapted to high-pressure deep-sea environments, making PBPRA2435 potentially relevant to pressure adaptation:

• Comparative genomics approaches:

  • Analysis of PBPRA2435 homologs across piezophilic and non-piezophilic bacteria

  • Identification of sequence variations correlated with depth adaptation

  • Evolutionary analysis to detect signatures of selection

• Experimental evolution studies:

  • Laboratory evolution of P. profundum under varying pressure conditions

  • Monitoring of PBPRA2435 sequence and expression changes

  • Targeted mutagenesis to test the role of specific residues

• Physiological studies:

  • Measurement of membrane physical properties (fluidity, thickness) as a function of pressure

  • Analysis of PBPRA2435 contribution to membrane adaptation

  • Investigation of potential role in pressure-sensing or response

• Photoreactivation and stress response connection:

  • Exploration of potential links between PBPRA2435 and UV damage repair pathways

  • Investigation of co-regulation with known stress response genes

  • Analysis of potential role in the photoreactivation process described for P. profundum

When studying PBPRA2435 in the context of environmental adaptation, experimental conditions should mimic the natural habitat of P. profundum, including appropriate pressure, temperature, salinity, and nutrient availability . This approach will provide the most physiologically relevant insights into protein function.