Recombinant Caulobacter crescentus UPF0391 membrane protein CC_0673 (CC_0673)

What is UPF0391 membrane protein CC_0673 and why is it studied?

UPF0391 membrane protein CC_0673 is a small 60-amino acid membrane-associated protein found in the aquatic bacterium Caulobacter crescentus. This protein belongs to the UPF (Uncharacterized Protein Family) 0391 classification, indicating that its function has not been fully characterized yet. Researchers study this protein to understand membrane organization in C. crescentus, which serves as a model organism for cell cycle regulation and survival in low-nutrient environments .

The amino acid sequence (MLKWAIILAIVALIAGALGFSGLAGAAAGVAKILFFLFLVGFVLVLLLGGTVFKAATGPK) suggests it contains hydrophobic regions consistent with membrane insertion, making it potentially important for membrane integrity or specialized membrane functions .

How does CC_0673 compare to other membrane proteins in C. crescentus?

Unlike the well-characterized outer membrane proteins in C. crescentus such as RsaFa and RsaFb (involved in S-layer export) or OmpA2 (involved in stalk growth and outer membrane stability), CC_0673's function remains largely uncharacterized . While RsaFa and RsaFb form part of a type I secretion system and share homology with E. coli's TolC protein, CC_0673 belongs to a different protein family .

OmpA2 shows a concentration gradient from stalk to pole and plays a role in membrane stability, while CC_0673's localization pattern remains to be determined . The small size of CC_0673 (60 amino acids) compared to OmpA2 suggests they likely have distinct functions in membrane organization.

What expression systems are recommended for CC_0673?

For recombinant expression of CC_0673, E. coli is the recommended expression system based on successful production of the His-tagged full-length protein . The methodology involves:

• Cloning the CC_0673 gene into an expression vector with an N-terminal His-tag

• Transforming into an E. coli expression strain

• Inducing protein expression (likely with IPTG, though specific conditions must be optimized)

• Purifying using nickel affinity chromatography

• Lyophilizing the purified protein for storage

As with other C. crescentus membrane proteins, expression in native C. crescentus may also be possible through electroporation of plasmid constructs followed by antibiotic selection and sucrose counterselection, similar to methods used for other membrane proteins in this organism .

What are the physicochemical properties of CC_0673?

The high proportion of hydrophobic residues (A, I, L, V, F, G) indicates strong membrane association, consistent with its classification as a membrane protein .

What structural insights exist for CC_0673?

Currently, no high-resolution structural data (X-ray crystallography or NMR) appears to be available for CC_0673. Structural predictions can be made using bioinformatic approaches:

• Secondary structure prediction suggests a predominantly alpha-helical structure

• Hydropathy analysis indicates potential transmembrane segments

• Homology modeling may provide limited insights, but the UPF0391 family has few characterized structural homologs

For experimental structure determination, researchers should consider:

• Detergent screening to identify optimal solubilizing conditions

• Crystallization trials with membrane-protein specific screens

• NMR spectroscopy for solution structure of detergent-solubilized protein

• Cryo-EM for potential structural analysis in membrane mimetics

How should CC_0673 be stored to maintain stability?

For optimal stability, store recombinant CC_0673 following these guidelines :

• Short-term storage: Store working aliquots at 4°C for up to one week

• Long-term storage: Store at -20°C or -80°C as a lyophilized powder

• Reconstitution:

  • Centrifuge vial briefly before opening

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

  • Add glycerol to 5-50% final concentration (50% recommended)

  • Aliquot for long-term storage

• Avoid: Repeated freeze-thaw cycles which can lead to protein degradation and aggregation

These storage recommendations are consistent with general practices for membrane proteins to prevent denaturation and loss of native conformation .

What are the optimal conditions for expressing recombinant CC_0673?

While specific optimization data for CC_0673 expression is not provided in the search results, successful expression strategies can be based on established protocols for other membrane proteins in C. crescentus:

• Vector selection: pET-based vectors with N-terminal His-tag appear successful

• E. coli strain: BL21(DE3) or Rosetta strains are commonly used for membrane proteins

• Induction parameters:

  • Temperature: Lowering to 18-25°C often improves membrane protein folding

  • Inducer concentration: 0.1-0.5 mM IPTG typically suitable

  • Duration: 4-16 hours (overnight) induction often balances yield and quality

• Media enrichment:

  • Addition of glucose (0.5-1%) may help repress basal expression

  • Addition of glycerol (0.5-1%) can support membrane protein expression

Experimental design should include a small-scale expression test varying these parameters before proceeding to larger-scale production.

What purification strategy is recommended for CC_0673?

Based on the recombinant His-tagged CC_0673 specifications, a purification strategy would include :

• Cell lysis:

  • Mechanical disruption (sonication, French press, or homogenization)

  • Buffer containing mild detergents (e.g., n-dodecyl-β-D-maltoside or CHAPS)

  • Protease inhibitors to prevent degradation

• Initial purification:

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

  • Washing with increasing imidazole concentrations (10-40 mM)

  • Elution with high imidazole (250-500 mM)

• Secondary purification:

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography if higher purity is required

• Final processing:

  • Buffer exchange to remove imidazole

  • Concentration using membrane filtration (10 kDa cutoff)

  • Lyophilization in Tris/PBS-based buffer with 6% trehalose, pH 8.0

How can researchers confirm successful expression and purification?

Multiple validation methods should be employed to confirm expression and purity:

• SDS-PAGE:

  • Use Tricine-SDS-PAGE systems optimized for small proteins

  • Expected molecular weight approximately 7-8 kDa (including His-tag)

  • Target purity > 90% as indicated in product specifications

• Western blotting:

  • Anti-His antibody detection

  • If available, specific antibodies against CC_0673

• Mass spectrometry:

  • MALDI-TOF MS to confirm molecular weight

  • LC-MS/MS for peptide identification and sequence verification

• Functional assays:

  • Membrane incorporation tests in liposomes

  • Potential protein-interaction studies

What methods can be used to study CC_0673 localization in bacterial cells?

To investigate cellular localization of CC_0673, researchers can employ techniques similar to those used for other C. crescentus membrane proteins like OmpA2 :

• Fluorescent protein fusions:

  • Create C- or N-terminal fusions with fluorescent proteins (e.g., GFP, mCherry)

  • Visualize using fluorescence microscopy to determine subcellular distribution

  • Compare localization patterns across the cell cycle

• Cell fractionation:

  • Separate cytoplasmic, inner membrane, periplasmic, and outer membrane fractions

  • Detect CC_0673 in fractions using Western blotting

  • Quantify relative distribution across cellular compartments

• Immunogold electron microscopy:

  • Develop specific antibodies against CC_0673

  • Visualize localization at nanometer resolution

  • Determine precise membrane association patterns

• Genetic position effects:

  • As observed with OmpA2, chromosome position may affect protein localization

  • Test expression from different chromosomal positions

  • Evaluate localization patterns to determine if similar position-dependent patterns exist

How can researchers identify potential interaction partners of CC_0673?

Several complementary approaches can identify protein-protein interactions:

• Pull-down assays:

  • Use His-tagged CC_0673 as bait

  • Cross-link proteins in native membranes before solubilization

  • Identify co-purifying proteins by mass spectrometry

• Bacterial two-hybrid screening:

  • Create fusion constructs with split reporter proteins

  • Screen against C. crescentus genomic library

  • Validate positive interactions with secondary assays

• Chemical cross-linking:

  • Apply membrane-permeable cross-linkers to intact cells

  • Identify cross-linked complexes by mass spectrometry

  • Map interaction surfaces through mutational analysis

• Co-immunoprecipitation:

  • Express epitope-tagged CC_0673 in C. crescentus

  • Precipitate protein complexes using specific antibodies

  • Identify co-precipitating proteins by mass spectrometry

What approaches can determine if CC_0673 is essential for C. crescentus viability?

To evaluate the essentiality of CC_0673:

• Gene deletion attempts:

  • Create knockout constructs using homologous recombination

  • Use counterselection systems (e.g., sucrose sensitivity with sacB gene)

  • Inability to obtain viable knockouts suggests essentiality

• Conditional expression systems:

  • Place CC_0673 under control of inducible promoters

  • Monitor growth and viability upon depletion

  • Quantify morphological changes during depletion

• Transposon mutagenesis screening:

  • Perform Tn-seq (transposon mutagenesis with deep sequencing)

  • Analyze insertion distribution patterns

  • Absence of insertions suggests essentiality, similar to analysis done for RsaFa and RsaFb genes

• Complementation testing:

  • Express CC_0673 from a plasmid

  • Attempt chromosomal deletion in the presence of plasmid-expressed protein

  • Test complementation with mutant versions to identify critical residues

How might CC_0673 relate to membrane protein translocation systems in C. crescentus?

Given C. crescentus' unique membrane protein export systems, CC_0673 could potentially play a role in:

• S-layer protein export:

  • C. crescentus utilizes RsaFa and RsaFb (TolC homologs) for S-layer export

  • CC_0673 might function in alternative export pathways or in modulating existing ones

  • Comparative studies with RsaFa/RsaFb mutants could reveal functional relationships

• Membrane integrity maintenance:

  • Similar to OmpA2's role in outer membrane stability

  • CC_0673 might contribute to specific membrane domains or structural integrity

  • Sensitivity testing to membrane-disrupting agents could reveal such functions

• Contact-dependent interaction systems:

  • C. crescentus utilizes Cdz system for contact-dependent killing

  • CC_0673 could potentially modulate cell-cell interactions or contact-dependent processes

  • Co-localization studies with Cdz components might reveal functional connections

How can researchers investigate potential roles in antimicrobial resistance?

Several C. crescentus membrane proteins contribute to antimicrobial resistance, suggesting potential similar roles for CC_0673:

• Comparative resistance profiling:

  • Create CC_0673 overexpression and depletion strains

  • Test minimum inhibitory concentrations against diverse antibiotics

  • Compare with known resistance determinants like RsaFa/RsaFb

• Membrane permeability assays:

  • Measure uptake of fluorescent dyes (e.g., propidium iodide, ethidium bromide)

  • Quantify changes in permeability upon CC_0673 depletion or overexpression

  • Compare results with RsaFa/RsaFb mutants, which show altered membrane permeability

• Outer membrane vesicle analysis:

  • Isolate outer membrane vesicles (OMVs) from wild-type and CC_0673 mutant strains

  • Compare protein and lipid composition

  • Evaluate potential changes in OMV production rates

What are the methodological challenges in studying small membrane proteins like CC_0673?

Researchers face specific challenges when working with small membrane proteins:

• Detection limitations:

  • Small size (60 amino acids) makes CC_0673 difficult to visualize on standard SDS-PAGE

  • Solution: Use specialized tricine-based gel systems optimized for small proteins

  • Alternative: Use epitope tags (His, FLAG) to enhance detection sensitivity

• Structural determination barriers:

  • Small membrane proteins often fail to crystallize using standard approaches

  • Solution: Consider specialized crystallization techniques like lipidic cubic phase

  • Alternative: Solid-state NMR for structure determination in membrane-mimetic environments

• Functional redundancy:

  • Small membrane proteins often exist in families with functional overlap

  • Solution: Create multiple knockout combinations to overcome redundancy

  • Example: RsaFa and RsaFb in C. crescentus show redundancy in S-layer export

• Native expression levels:

  • Small membrane proteins are often expressed at low levels

  • Solution: Develop highly sensitive detection methods (e.g., targeted mass spectrometry)

  • Alternative: Create reporter fusions that amplify detection signal without disrupting function

Are there homologs of CC_0673 in other bacterial species?

While specific homology data for CC_0673 is not provided in the search results, researchers can investigate evolutionary relationships through:

• Sequence similarity searches:

  • BLAST against diverse bacterial genomes

  • Focus on alpha-proteobacteria and other aquatic bacteria

  • Identify conserved sequence motifs across homologs

• Phylogenetic analysis:

  • Construct phylogenetic trees of UPF0391 family proteins

  • Map presence/absence patterns across bacterial taxonomy

  • Correlate with habitat and physiological adaptations

• Genomic context analysis:

  • Examine gene neighborhoods around CC_0673 homologs

  • Identify conserved operonic structures

  • Infer potential functional associations from genomic context

This evolutionary perspective may reveal important insights about the function and significance of this small membrane protein across different bacterial lineages.

How do experimental approaches differ between studying CC_0673 in C. crescentus versus heterologous systems?

Researchers should consider these differences when designing experiments and interpreting results from different expression systems.