Recombinant Listeria welshimeri serovar 6b UPF0754 membrane protein lwe2241 (lwe2241)

What are the predicted structural characteristics of lwe2241 protein based on its sequence?

Based on the amino acid sequence, lwe2241 is predicted to be a membrane protein with multiple transmembrane domains. The high content of hydrophobic residues (particularly in the N-terminal and C-terminal regions) suggests a typical membrane protein topology. Computational tools predict that this protein likely contains multiple alpha-helical transmembrane segments, consistent with its classification as a UPF0754 family membrane protein.

Advanced structural analysis requires experimental methods such as X-ray crystallography or cryo-electron microscopy, but preliminary structure prediction can be performed using AI-based tools like AlphaFold2, which have significantly improved protein structure prediction accuracy .

How does the expression system affect the yield and functionality of recombinant lwe2241 protein?

The expression system significantly impacts both yield and functionality of recombinant lwe2241. As a membrane protein, lwe2241 presents particular challenges:

E. coli expression system: The standard approach used for the His-tagged recombinant lwe2241 provides reasonable yields but may face challenges with proper folding and insertion of transmembrane domains . E. coli systems typically require optimization of:

• Induction temperature (often lowered to 16-25°C)

• Inducer concentration

• Expression time

• Specialized E. coli strains designed for membrane proteins

Alternative expression systems: For improved functionality, especially if the protein requires post-translational modifications, eukaryotic systems might be considered:

• Yeast (Pichia pastoris, Saccharomyces cerevisiae)

• Insect cells (Sf9, High Five)

• Mammalian cells (CHO, HEK293)

Each system presents a trade-off between yield, functionality, and experimental complexity. For structural studies requiring high yields, E. coli remains the most commonly used system despite potential folding challenges .

What strategies can optimize the expression of this transmembrane protein in E. coli?

Optimizing expression of lwe2241 in E. coli requires addressing several challenges specific to membrane proteins:

Codon optimization: Analyzing the lwe2241 sequence for rare codons and optimizing them for E. coli expression can significantly improve translation efficiency. This is particularly important for proteins from non-E. coli bacterial sources like Listeria welshimeri .

Expression vector selection: For membrane proteins like lwe2241, vectors with tightly controlled promoters (such as T7lac or araBAD) allow fine-tuning of expression levels to prevent toxic accumulation. The His-tag placement (N-terminal in the characterized construct) allows for easy purification while minimizing interference with transmembrane domain insertion .

Culture conditions optimization:

• Lower induction temperatures (16-25°C) slow down expression rate, allowing proper folding and membrane insertion

• Reduced inducer concentrations

• Extended expression time (24-72 hours)

• Addition of membrane-stabilizing compounds (glycerol, specific detergents)

Specialized E. coli strains:

• C41(DE3) and C43(DE3): Specifically engineered for membrane protein expression

• Lemo21(DE3): Allows tunable expression levels

• Rosetta strains: Provide rare tRNAs that may be required for efficient translation

These approaches can be used individually or in combination to overcome expression challenges for transmembrane proteins like lwe2241 .

What purification protocol yields the highest purity and activity for recombinant lwe2241 protein?

A high-yield purification protocol for recombinant His-tagged lwe2241 membrane protein involves:

Membrane extraction and solubilization:

• Cell lysis using mechanical disruption (sonication or high-pressure homogenization)

• Isolation of membrane fraction by ultracentrifugation

• Membrane solubilization using appropriate detergents:

  • Initial screening of detergents (DDM, LDAO, CHAPS, OG)

  • Optimal detergent concentration determination

  • Incubation time optimization (typically 1-3 hours at 4°C)

Affinity chromatography:

• Ni-NTA chromatography using the His-tag

• Careful washing with increasing imidazole concentrations to remove non-specific binding

• Elution with high imidazole (250-500 mM)

Secondary purification:

• Size exclusion chromatography to separate aggregates and ensure homogeneity

• Optional ion exchange chromatography for further purification

Quality control:

• SDS-PAGE analysis showing >90% purity

• Western blot confirmation

• Mass spectrometry verification

This protocol has been shown to yield protein with greater than 90% purity as determined by SDS-PAGE , which is suitable for most structural and functional studies.

How can researchers determine if the purified lwe2241 protein maintains its native conformation?

Verifying native conformation of purified lwe2241 is essential for functional studies. Multiple complementary techniques should be employed:

Circular Dichroism (CD) Spectroscopy:

• Far-UV CD (190-260 nm) to assess secondary structure content

• Near-UV CD (250-350 nm) to evaluate tertiary structure integrity

• Thermal denaturation studies to determine stability

Fluorescence Spectroscopy:

• Intrinsic tryptophan fluorescence to monitor tertiary structure

• Binding of structure-sensitive fluorescent probes

Functional Assays:

• Binding studies with known interaction partners

• Enzymatic activity assays if applicable

Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS):

• Confirms protein is monodisperse and not aggregated

• Determines oligomeric state in solution

Transmission Electron Microscopy (negative staining):

• Visual confirmation of proper folding

• Assessment of homogeneity

For lwe2241 specifically, researchers should consider detergent screening using these techniques to identify conditions that best maintain native conformation after purification .

What crystallization conditions have been successful for membrane proteins similar to lwe2241?

While specific crystallization conditions for lwe2241 have not been reported in the provided search results, successful approaches for similar bacterial membrane proteins include:

Detergent-based crystallization:

• Initial detergent screening using DDM, DM, NG, LMNG, and OG

• Detergent concentration optimization (typically 1-3× CMC)

• Addition of lipids (0.1-0.5 mg/ml) to stabilize the protein

• Crystallization at temperatures between 4-20°C

Lipid cubic phase (LCP) crystallization:

• Reconstitution in monoolein or other lipid matrices

• Screening with different precipitants and additives

• Optimization of protein:lipid ratios (typically 2:3 w/w)

• Extended incubation times (weeks to months)

Bicelle-based approaches:

• Using DMPC/CHAPSO bicelles at ratios of 2.8-3.2

• Protein reconstitution at various protein:bicelle ratios

• Temperature cycling between 4°C and 20°C

Successful crystallization typically requires extensive screening (500-1000 conditions) and optimization of promising initial hits .

How can researchers apply cryo-electron microscopy to study the structure of lwe2241?

Cryo-electron microscopy (cryo-EM) has emerged as a powerful technique for membrane protein structural studies and could be applied to lwe2241 using the following approach:

Sample preparation:

• Purification in suitable detergents (DDM, LMNG) or reconstitution into nanodiscs/liposomes

• Concentration optimization (typically 2-5 mg/ml)

• Grid preparation using holey carbon films

• Vitrification by plunge-freezing in liquid ethane

Data collection strategy:

• Microscope selection (300 kV preferred for high resolution)

• Detector selection (direct electron detectors)

• Collection parameters optimization (defocus range, exposure)

• Motion correction and CTF estimation

Data processing workflow:

• Particle picking (manual or automated)

• 2D classification to select well-defined classes

• Initial model generation

• 3D classification and refinement

• Post-processing and validation

For membrane proteins like lwe2241, cryo-EM offers advantages over crystallography, particularly for proteins that are challenging to crystallize. The use of nanodiscs or amphipols can help maintain the protein in a native-like lipid environment during analysis .

What computational approaches can predict functional domains in lwe2241 without experimental structural data?

In the absence of experimental structural data, several computational approaches can provide valuable insights into lwe2241 functional domains:

Sequence-based prediction tools:

• TMHMM, HMMTOP for transmembrane domain prediction

• SignalP for signal peptide identification

• InterProScan for conserved domain detection

• Pfam and SMART databases for functional domain annotation

Evolutionary analysis:

• Multiple sequence alignment with homologous proteins

• Conservation analysis to identify functionally important residues

• Coevolution analysis to predict residue interactions

AI-based structure prediction:

• AlphaFold2 or RoseTTAFold for 3D structure prediction

• Validation of predicted structures using ProSA, PROCHECK

• Mapping of conserved residues onto predicted structures

Molecular dynamics simulations:

• Assessment of stability in membrane environments

• Prediction of potential binding sites

• Investigation of conformational flexibility

These approaches can be used synergistically to develop testable hypotheses about lwe2241 function that can guide experimental design .

What experimental approaches can determine the membrane topology of lwe2241?

Determining the membrane topology of lwe2241 is crucial for understanding its function. Several complementary approaches can be employed:

Cysteine accessibility methods:

• Introduction of cysteine residues at various positions

• Treatment with membrane-permeable and -impermeable sulfhydryl reagents

• Detection of modification using mass spectrometry or fluorescent labeling

Fusion protein approaches:

• C-terminal and N-terminal fusions with reporter proteins (GFP, alkaline phosphatase, β-lactamase)

• Analysis of reporter activity to determine orientation

• Systematic construction of truncation-fusion proteins

Protease protection assays:

• Treatment of membrane preparations with proteases

• Western blot analysis with antibodies against different regions

• Mass spectrometry identification of protected fragments

Fluorescence quenching:

• Introduction of fluorescent probes at specific positions

• Analysis of quenching by membrane-impermeable agents

• Determination of residue accessibility

A comprehensive topology map typically requires combining multiple approaches to resolve ambiguities, particularly for proteins with multiple transmembrane segments like lwe2241 .

How can researchers identify potential interaction partners of lwe2241?

Identifying interaction partners provides crucial insights into lwe2241 function. Several approaches can be employed:

Affinity-based methods:

• Pull-down assays using His-tagged lwe2241 as bait

• Co-immunoprecipitation with anti-lwe2241 antibodies

• Tandem affinity purification followed by mass spectrometry

Proximity labeling approaches:

• BioID or TurboID fusion proteins

• APEX2 fusion for proximity-dependent biotinylation

• Mass spectrometry identification of labeled proteins

Genetic approaches:

• Bacterial two-hybrid screening

• Synthetic genetic array analysis

• Suppressor mutant screening

Crosslinking studies:

• Chemical crosslinking with membrane-permeable reagents

• Photo-crosslinking with unnatural amino acids

• Mass spectrometry analysis of crosslinked products

Computational prediction:

• Interactome database mining

• Coevolution analysis

• Structural docking simulations

These approaches can be used sequentially, starting with computational predictions to guide experimental design, followed by in vitro validation and in vivo confirmation of physiologically relevant interactions .

What methods can determine if lwe2241 forms functional oligomers?

Determining the oligomeric state of lwe2241 is important for understanding its functional mechanism. Several techniques can provide this information:

Analytical ultracentrifugation:

• Sedimentation velocity experiments to determine size distribution

• Sedimentation equilibrium for accurate molecular weight determination

• Analysis in various detergent conditions to assess detergent effects

Size exclusion chromatography with multi-angle light scattering (SEC-MALS):

• Determination of absolute molecular weight independent of shape

• Assessment of homogeneity and polydispersity

• Analysis of detergent contribution to measured mass

Chemical crosslinking:

• Treatment with various crosslinkers (DSS, BS3, glutaraldehyde)

• SDS-PAGE analysis of crosslinked products

• Mass spectrometry for crosslink identification

Native mass spectrometry:

• Analysis of intact membrane protein complexes

• Determination of subunit stoichiometry

• Detection of bound lipids or cofactors

Single-molecule methods:

• Fluorescence resonance energy transfer (FRET)

• Single-molecule photobleaching

• Sub-stoichiometric labeling

These techniques provide complementary information and should be used in combination to establish the oligomeric state of lwe2241 under physiologically relevant conditions .

How can recombinant lwe2241 protein be used in structural biology studies?

Recombinant lwe2241 protein serves as a valuable tool for structural biology research through multiple approaches:

X-ray crystallography:

• Purified His-tagged lwe2241 can be used for crystallization trials

• Structure determination at atomic resolution

• Co-crystallization with ligands or interaction partners

Cryo-electron microscopy:

• Single-particle analysis for high-resolution structure determination

• Visualization in different functional states

• Structure of lwe2241 in complex with binding partners

Nuclear magnetic resonance (NMR) spectroscopy:

• Solution NMR for dynamic regions or smaller domains

• Solid-state NMR for full-length protein in membrane mimetics

• Investigation of protein-ligand interactions

Small-angle X-ray scattering (SAXS):

• Low-resolution envelope determination

• Analysis of conformational changes

• Validation of computational models

The high purity of recombinant lwe2241 (>90% as determined by SDS-PAGE) makes it suitable for these structural biology applications, which require homogeneous protein preparations .

What experimental design would best characterize the role of lwe2241 in Listeria welshimeri?

A comprehensive experimental design to characterize lwe2241's role in Listeria welshimeri would include:

Genetic approaches:

• Construction of lwe2241 deletion mutant

• Phenotypic characterization:

  • Growth curves in various conditions

  • Membrane integrity assays

  • Stress response profiling

• Complementation studies with wild-type and mutant variants

Localization studies:

• Fluorescent protein fusions to determine subcellular localization

• Immunogold electron microscopy for high-resolution localization

• Fractionation studies to confirm membrane association

Comparative genomics:

• Analysis of lwe2241 conservation across Listeria species

• Identification of co-evolving genes

• Prediction of functional partners

Transcriptomic and proteomic analyses:

• RNA-seq of wild-type vs. deletion mutant

• Proteome analysis to identify altered protein expression

• Phosphoproteomics to detect signaling changes

Biochemical characterization:

• Purification of recombinant lwe2241

• Functional assays based on bioinformatic predictions

• Interaction studies with candidate partners

This multi-faceted approach would provide comprehensive insights into lwe2241 function within the bacterial cell .

How can researchers overcome low expression yields of recombinant lwe2241?

Low expression yields are common with membrane proteins like lwe2241. Several strategies can address this challenge:

Expression system optimization:

• Testing multiple expression systems:

  • Different E. coli strains (BL21, C41/C43, Rosetta)

  • Alternative hosts (yeast, insect cells)

• Vector optimization:

  • Promoter strength adjustment

  • Codon optimization for host

  • Fusion tags (MBP, SUMO) to enhance solubility

Expression condition optimization:

• Temperature reduction (16-25°C)

• Induction at different growth phases

• Extended expression time with reduced inducer concentration

• Addition of chemical chaperones (glycerol, betaine, sorbitol)

Cell-free expression systems:

• E. coli extract-based systems with added detergents or lipids

• Direct expression into nanodiscs or liposomes

• Continuous-exchange cell-free systems for higher yields

Fusion partner approaches:

• N-terminal fusions that enhance expression (MBP, GST, SUMO)

• C-terminal stability-enhancing tags

• Systematic testing of tag positions and linker lengths

For lwe2241 specifically, expression in specialized E. coli strains at reduced temperatures (16-20°C) with extended induction times (16-24 hours) typically provides significant improvement in yields .

What strategies can address protein aggregation during lwe2241 purification?

Membrane protein aggregation during purification is a common challenge that can be addressed through multiple strategies:

Detergent optimization:

• Systematic screening of detergents:

  • Harsh (SDS, LDAO)

  • Mild (DDM, LMNG, GDN)

  • Zwitterionic (CHAPS, Fos-choline)

• Testing detergent mixtures

• Gradual detergent exchange during purification

Stabilizing additives:

• Glycerol (10-20%)

• Specific lipids (cholesterol, DOPE, POPG)

• Osmolytes (sucrose, trehalose)

• Specific ligands or binding partners

Purification conditions:

• Temperature control (typically 4°C throughout)

• Addition of reducing agents (DTT, TCEP)

• Optimization of pH and ionic strength

• Use of stabilizing buffer components

Alternative solubilization approaches:

• Amphipols

• Nanodiscs

• SMALPs (styrene-maleic acid lipid particles)

• Saposin-lipoprotein nanoparticles

For recombinant lwe2241, purification in the presence of 6% trehalose has been shown to improve stability, as indicated in the storage buffer recommendations .

How can researchers validate the functionality of purified recombinant lwe2241?

Validating functionality of purified lwe2241 requires multiple approaches:

Structural integrity assessment:

• Circular dichroism to confirm secondary structure

• Fluorescence spectroscopy to assess tertiary structure

• Thermal stability assays (differential scanning fluorimetry)

Binding assays:

• Surface plasmon resonance with predicted ligands

• Microscale thermophoresis for interaction studies

• Isothermal titration calorimetry for thermodynamic parameters

Functional reconstitution:

• Liposome reconstitution

• Proteoliposome-based functional assays

• Planar lipid bilayer experiments if channel activity is suspected

In vitro complementation:

• Addition of purified protein to membrane preparations from knockout strains

• Rescue of specific biochemical activities

• Competitive binding assays with native protein

Comparative analysis:

• Parallel characterization of wild-type and mutant variants

• Activity comparison with homologous proteins

• Structure-function correlation studies

These validation approaches ensure that the purified recombinant lwe2241 retains its native functional properties despite the potential stresses of expression and purification .

How might lwe2241 function compare across different Listeria species?

Understanding lwe2241 function across Listeria species requires comparative analysis:

Sequence analysis:

• Multiple sequence alignment of lwe2241 homologs

• Identification of conserved and variable regions

• Evolutionary rate analysis to detect selection pressures

Phylogenetic profiling:

• Construction of phylogenetic trees

• Correlation with species-specific traits

• Identification of co-evolving genes

Heterologous expression:

• Expression of homologs from different species

• Functional complementation tests

• Comparative biochemical characterization

Structural comparison:

• Homology modeling of different homologs

• Identification of structural differences

• Correlation of structural variations with functional differences

Genomic context analysis:

• Comparison of operon organization

• Analysis of regulatory elements

• Identification of species-specific genetic associations

This comparative approach can reveal the evolutionary conservation of lwe2241 function or identify species-specific adaptations that may be related to ecological niches or pathogenicity .

What protein engineering approaches could enhance lwe2241 stability for structural studies?

Enhancing lwe2241 stability through protein engineering can significantly improve success in structural studies:

Targeted mutagenesis:

• Surface entropy reduction (replacement of flexible, charged residues)

• Introduction of disulfide bonds at strategic positions

• Proline substitutions in loop regions

• Glycine to alanine mutations to reduce flexibility

Domain truncation and fusion:

• Removal of flexible termini or loops

• Construction of minimal functional domains

• Fusion with crystallization chaperones (T4 lysozyme, BRIL)

• Addition of thermostabilizing domains

Directed evolution:

• Random mutagenesis and screening for enhanced stability

• Error-prone PCR followed by expression screening

• Yeast display selection for stable variants

• Phage display with stability selection pressure

Computational design:

• In silico identification of destabilizing residues

• Energy minimization through computational modeling

• Prediction and design of stabilizing interactions

• Consensus-based design from multiple homologs

Chimera construction:

• Grafting stable regions from homologous proteins

• Domain swapping with well-characterized homologs

• Introduction of stabilizing motifs from thermophilic organisms

These approaches can be applied iteratively, with each round of engineering followed by stability assessment to gradually improve lwe2241 properties for structural studies .

What role might lwe2241 play in bacterial membrane biology based on sequence homology?

Analysis of lwe2241 sequence homology provides insights into its potential role in bacterial membrane biology:

Functional prediction from homology:

• UPF0754 family proteins are conserved across many bacterial species

• Predicted membrane localization suggests involvement in:

  • Membrane integrity maintenance

  • Transport processes

  • Signal transduction

  • Cell envelope biogenesis

Structural homology:

• Alpha-helical transmembrane domains suggest a channel, transporter, or receptor function

• Structural similarities with characterized bacterial membrane proteins can indicate functional parallels

Domain architecture analysis:

• Identification of conserved motifs associated with specific functions

• Recognition of functional domains through distant homology

• Prediction of binding sites or catalytic residues

Genomic context clues:

• Co-occurrence with genes of known function

• Operon structure analysis

• Conservation of genomic neighborhood across species

Based on these analyses, lwe2241 likely plays a role in membrane processes essential for bacterial cell function, possibly related to transport, signaling, or membrane organization. Further experimental characterization is needed to confirm these predictions and elucidate the specific molecular mechanisms involved .