Recombinant UPF0197 transmembrane protein CBG19073 (CBG19073)

What is the basic structure of UPF0197 transmembrane protein CBG19073?

UPF0197 transmembrane protein CBG19073 is a 79 amino acid transmembrane protein from Caenorhabditis briggsae with a molecular weight of 8,692 Da. The protein sequence includes hydrophobic regions consistent with a transmembrane domain, specifically: MDISKMDRYA APVHFSSLPL LATVLCGVGL LLLAAFTMLQ VTSTKYNRNV FKELFIAATS SIFLGFGSVF LLLWVGIYV. The sequence suggests a single-span transmembrane protein with hydrophobic residues concentrated in the membrane-spanning region .

What is the predicted membrane topology of CBG19073?

Based on structural analysis, CBG19073 likely adopts a single transmembrane helix conformation. The protein contains a hydrophobic core region (residues approximately 20-45) that spans the membrane, with flanking hydrophilic regions that extend into the cytoplasmic and extracellular spaces. This topology is consistent with other members of the UPF0197 family, which typically feature a single membrane-spanning domain with specific amino acid positioning that facilitates interaction with membrane lipids .

What expression systems are optimal for producing recombinant CBG19073?

For CBG19073, cell-free expression systems have proven particularly effective, allowing the production of functional transmembrane protein without the complications associated with membrane protein overexpression in cellular systems. This approach circumvents issues like protein aggregation, misfolding, or cytotoxicity that often occur when expressing transmembrane proteins in bacterial or eukaryotic cell cultures. The commercially available recombinant CBG19073 achieves ≥85% purity using this cell-free expression method .

What are the recommended storage conditions for maintaining CBG19073 stability?

Recombinant CBG19073 should be stored at -20°C for regular use, or at -80°C for extended storage periods. The protein is typically supplied in a solution containing glycerol as a cryoprotectant. Repeated freeze-thaw cycles should be avoided to prevent protein degradation. For working aliquots, storage at 4°C is recommended for up to one week. When receiving the protein, brief centrifugation is advised if liquid becomes entrapped in the vial cap during shipment .

What purification approaches yield the highest quality CBG19073 preparations?

High-purity CBG19073 preparations (≥85%) are typically achieved through a combination of affinity chromatography and size exclusion techniques. SDS-PAGE analysis is the standard method for assessing purity. For transmembrane proteins like CBG19073, the addition of appropriate detergents during purification is critical to maintain native conformation and prevent aggregation. The specific detergent selection should be optimized based on downstream applications, with milder detergents preferred for functional studies .

What experimental approaches can reveal CBG19073's functional roles in membrane biology?

To elucidate CBG19073's functional roles, researchers should implement a multi-faceted approach:

• Protein-protein interaction studies: Techniques like co-immunoprecipitation, proximity labeling (BioID, APEX), or FRET can identify binding partners within membrane complexes.

• Lipid interaction analysis: Lipid binding assays, including liposome flotation assays and surface plasmon resonance with lipid nanodiscs, can identify specific lipid preferences.

• Localization studies: Super-resolution microscopy combined with fluorescently-tagged constructs can reveal subcellular distribution patterns and potential co-localization with functional membrane domains.

• Loss-of-function analysis: CRISPR-Cas9 mediated knockout or knockdown approaches in C. briggsae, followed by phenotypic characterization, can illuminate physiological roles.

Integration of these methodologies provides complementary insights into transmembrane protein function beyond what single approaches might reveal .

How can researchers study potential protein-protein interactions involving CBG19073?

To investigate protein-protein interactions for CBG19073, researchers should consider specialized approaches for transmembrane proteins:

• Membrane yeast two-hybrid systems: Modified Y2H systems designed specifically for membrane proteins can overcome limitations of traditional systems.

• Chemical crosslinking coupled with mass spectrometry: This approach captures transient interactions in native membrane environments.

• Single-molecule tracking: This technique can detect co-diffusion of CBG19073 with potential interaction partners in live cell membranes.

• Computational prediction followed by experimental validation: Molecular modeling of CBG19073's transmembrane domain can identify potential interaction motifs for targeted mutagenesis studies.

When designing these experiments, controls should include parallel analysis of transmembrane proteins with known interaction patterns to validate methodology efficacy .

What computational approaches aid in predicting CBG19073 interaction interfaces?

Advanced computational methods for predicting CBG19073 interaction interfaces include:

• Molecular dynamics simulations: All-atom or coarse-grained simulations of CBG19073 embedded in lipid bilayers can reveal conformational dynamics and potential interaction surfaces.

• Evolutionary coupling analysis: This approach identifies co-evolving residues that often represent functionally important interaction sites.

• Transmembrane helix-helix interaction prediction: Specialized algorithms like PREDDIMER or MEMPACK can identify potential homodimerization or heterodimerization interfaces.

• Integrative structural modeling: Combining low-resolution experimental data (such as crosslinking constraints) with computational modeling can generate testable structural hypotheses.

These computational predictions should guide targeted mutagenesis experiments to validate predicted interaction interfaces experimentally .

What are the critical considerations for reconstituting CBG19073 into artificial membrane systems?

Successful reconstitution of CBG19073 into artificial membrane systems requires careful optimization of multiple parameters:

The reconstitution process should be validated by assessing protein orientation using protease protection assays or antibody accessibility in intact proteoliposomes. Characterization of reconstituted systems should include dynamic light scattering and negative-stain EM to confirm vesicle size distribution and homogeneity .

What strategies can overcome challenges in structural studies of CBG19073?

Structural characterization of transmembrane proteins like CBG19073 presents unique challenges that can be addressed through specialized approaches:

• NMR spectroscopy optimizations:

  • Use of detergent micelles or lipid nanodiscs as membrane mimetics

  • Selective isotopic labeling to simplify spectral complexity

  • Solid-state NMR for proteins in lipid bilayers

• Cryo-EM considerations:

  • Reconstitution in nanodiscs or amphipols to improve particle distribution

  • Use of antibody fragments to increase protein mass

  • Implementation of specialized grid preparation techniques for membrane proteins

• X-ray crystallography approaches:

  • Lipidic cubic phase crystallization

  • Addition of stabilizing fusion proteins

  • Targeted surface mutations to enhance crystallizability without affecting structure

• Hybrid methods:

  • Integration of low-resolution data from small-angle X-ray scattering with computational modeling

  • Cross-validation between different structural techniques

Each approach has distinct advantages, with method selection depending on specific research questions and available resources .

How can researchers effectively design mutations to probe CBG19073 function?

Strategic mutation design for CBG19073 functional analysis should incorporate:

• Evolutionary conservation analysis: Target residues conserved across species, which often indicate functional importance.

• Transmembrane segment scanning: Systematic alanine or leucine scanning across the transmembrane domain to identify critical positioning residues.

• Charge manipulation experiments: Introduction or neutralization of charged residues at membrane interfaces to assess impact on topology and function.

• Domain swap approaches: Replacing segments with corresponding regions from related proteins to identify functional domains.

When analyzing mutant phenotypes, researchers should employ multiple complementary readouts:

This multi-parameter assessment ensures comprehensive characterization of mutant effects .

How can researchers apply de novo design principles to study CBG19073 interactions?

Recent advances in de novo transmembrane protein design can be applied to study CBG19073 through:

• Computational design of interaction partners: Using computational tools to design synthetic transmembrane peptides that selectively bind to CBG19073 in defined orientations. This approach has been successfully demonstrated with other transmembrane proteins, such as with the erythropoietin receptor (EpoR) transmembrane domain.

• Interface engineering: Designing specific amino acid contacts to probe potential binding interfaces of CBG19073, creating custom binding topologies.

• Competition assays: Developing synthetic transmembrane domains that compete with natural interaction partners to assess binding determinants and functional consequences.

• Structural validation: Using techniques like NMR to confirm that designed interactions occur through intended amino acid contacts and in predicted topologies.

This de novo design approach offers unprecedented precision in dissecting transmembrane protein interactions, moving beyond traditional mutagenesis strategies to custom-designed molecular tools .

What are the emerging methodologies for studying CBG19073 dynamics in membrane environments?

Cutting-edge approaches for investigating CBG19073 dynamics include:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) adapted for membrane proteins: This technique can reveal dynamic regions and conformational changes under different conditions.

• Single-molecule FRET (smFRET): By introducing fluorophore pairs at strategic positions, researchers can track real-time conformational changes of individual CBG19073 molecules.

• Native mass spectrometry: Recent adaptations for membrane proteins allow detection of intact complexes and their stoichiometry while preserving non-covalent interactions.

• High-speed atomic force microscopy (HS-AFM): This technique enables visualization of dynamic structural changes in membrane proteins at near-atomic resolution under physiological conditions.

• Time-resolved cryo-EM: Emerging methodologies capture structural transitions by rapidly freezing samples at defined time points after activation.

These approaches are particularly valuable for understanding how transmembrane proteins like CBG19073 respond to changes in membrane composition, interact with binding partners, or undergo conformational changes during function .

How might CBG19073 research inform broader transmembrane protein design strategies?

Studies of CBG19073 can contribute to broader transmembrane protein design principles through:

• Identification of minimal functional units: As a small transmembrane protein, CBG19073 may reveal fundamental principles about the minimal structural requirements for membrane integration and function.

• Sequence-structure relationships: Analysis of how specific sequence features determine membrane topology, stability, and interaction specificity can inform design rules.

• Environmental adaptation: Understanding how CBG19073 stability is maintained in different membrane environments can reveal principles for designing environment-responsive transmembrane domains.

• De novo design validation: CBG19073 can serve as a template for computational design algorithms, with successful prediction of its properties validating approaches that can be applied to design novel transmembrane proteins.

• Modular functionality: Identifying functional modules within CBG19073 that could be incorporated into synthetic biology applications or therapeutic design.

The insights gained from studying this relatively simple transmembrane protein can establish foundational principles for the rational design of more complex membrane-embedded systems with tailored properties and functions .

Future Research Directions

Future research on CBG19073 will likely benefit from emerging technologies in structural biology, computational modeling, and synthetic biology. Integration of de novo design approaches with traditional biochemical and biophysical techniques promises to reveal new insights into transmembrane protein structure-function relationships. As methodologies continue to advance, particularly in areas such as time-resolved structural biology and single-molecule analyses, our understanding of CBG19073 and related transmembrane proteins will expand, potentially informing applications in biotechnology and therapeutic design.