Recombinant Caulobacter crescentus UPF0060 membrane protein CC_1976 (CC_1976)

What is the basic structure of Caulobacter crescentus UPF0060 membrane protein CC_1976?

Caulobacter crescentus UPF0060 membrane protein CC_1976 is a full-length protein (typically 1-108 amino acids) belonging to the UPF0060 protein family. Similar to other characterized membrane proteins from this organism, it contains transmembrane domains that anchor it to the bacterial cell membrane. The protein plays a potential role in the asymmetric cell division cycle of C. crescentus, which is orchestrated by an elaborate gene-protein regulatory network .

When expressed recombinantly, the protein is often tagged with an N-terminal histidine tag to facilitate purification and is available as a lyophilized powder after expression and isolation .

What expression systems are most effective for producing recombinant CC_1976?

Based on successful protocols with similar Caulobacter membrane proteins, E. coli is the preferred heterologous expression system for CC_1976 . When designing expression vectors for optimal production, several factors should be considered:

• Translation initiation site accessibility is critical - opening energies less than or equal to 12 kcal/mol appear optimum for efficient protein production .

• Strong correlation exists between protein expression levels and the accessibility of the translation initiation site, which is a more significant factor than codon adaptation index (CAI) or tRNA adaptation index (tAI) .

• While higher accessibility leads to higher protein production, it may result in slower cell growth as demonstrated in stochastic simulation models .

The table below summarizes key parameters for optimizing recombinant CC_1976 expression:

What are the most effective purification strategies for recombinant His-tagged CC_1976?

Purification of His-tagged CC_1976 requires careful consideration of its membrane protein nature. The following methodology has proven effective for similar Caulobacter membrane proteins:

• Cell lysis: Gentle disruption using either sonication or cell disruptors in buffer containing mild detergents (e.g., n-dodecyl-β-D-maltoside or CHAPS) to solubilize membrane proteins without denaturation.

• Affinity chromatography: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin, with careful optimization of imidazole concentration in wash buffers (10-40 mM) to remove non-specifically bound proteins while retaining the His-tagged target protein.

• Size exclusion chromatography: Further purification to obtain monodisperse protein preparation suitable for structural and functional studies.

• Detergent exchange: If necessary for downstream applications, detergent exchange can be performed during size exclusion chromatography.

This multi-step approach typically yields protein with >90% purity suitable for downstream biochemical and structural analyses .

How can researchers assess the proper folding and stability of purified CC_1976?

Assessment of proper folding and stability is critical for membrane proteins like CC_1976. The following methodological approaches are recommended:

These techniques provide complementary information about protein folding and stability, which is essential for designing reliable functional assays .

What role does CC_1976 potentially play in the Caulobacter crescentus cell cycle?

Caulobacter crescentus has emerged as a model organism for studying asymmetric cell division and differentiation. While the specific function of CC_1976 is not fully characterized, its classification as a membrane protein suggests potential roles in:

• Cell envelope biogenesis or integrity maintenance during the asymmetric cell division cycle.

• Possible involvement in the elaborate gene-protein regulatory network centered on the three major control proteins (DnaA, GcrA, and CtrA) that orchestrate the C. crescentus cell cycle .

• Potential participation in the swarmer-to-stalked cell transition (differentiation), which involves several membrane proteins and the remodeling of the cell envelope.

Research into CC_1976 function could benefit from the computational models that have been developed for C. crescentus cell cycle regulation, which provide a framework for testing hypotheses about molecular mechanisms in silico .

How can researchers integrate CC_1976 studies into broader understanding of alpha-proteobacterial membrane proteins?

The cell cycle control proteins of Caulobacter are conserved across many species of alpha-proteobacteria, making CC_1976 research potentially applicable to other genera of importance to agriculture and medicine (e.g., Rhizobium, Brucella) . Integration strategies include:

• Comparative genomics and proteomics approaches to identify homologs in related bacterial species.

• Structural comparison with membrane proteins of similar function from related organisms to identify conserved domains or motifs.

• Development of heterologous expression systems in related alpha-proteobacteria to study function in a more native-like environment.

• Application of quantitative computational models, similar to those developed for C. crescentus cell cycle regulation, to investigate how membrane proteins like CC_1976 contribute to cellular physiology in different bacterial species.

This integrative approach can provide insights into the evolution and conservation of membrane protein functions across the alpha-proteobacteria .

What are the optimal conditions for crystallization trials of CC_1976?

Crystallization of membrane proteins remains challenging but is essential for high-resolution structural determination. For CC_1976, consider the following methodological approach:

• Detergent screening: Test a panel of detergents (maltoside series, glucoside series, neopentyl glycol derivatives) for protein stability and monodispersity using SEC-MALS before initiating crystallization trials.

• Lipid cubic phase (LCP) crystallization: Often more successful for membrane proteins than vapor diffusion methods. Use monoolein or other lipids mixed with purified protein in detergent.

• Addition of lipids: Supplementing crystallization trials with specific phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine) can stabilize membrane proteins and promote crystal contacts.

• Protein engineering: Consider designing constructs with truncated termini or loop regions to reduce conformational heterogeneity, or fusion proteins (e.g., T4 lysozyme fusion) to increase soluble surface area for crystal contacts.

• Automated crystallization screening: Use high-throughput robotic systems to test hundreds of conditions with minimal protein consumption.

Success in crystallization requires iterative optimization based on initial screening results, with careful attention to protein purity, homogeneity, and stability .

How can advanced computational modeling inform experimental design for CC_1976 research?

Computational modeling can substantially enhance experimental research on CC_1976 through:

• Molecular dynamics simulations: To predict protein behavior in membrane environments and identify potential conformational changes relevant to function.

• Homology modeling: If structural data for close homologs exist, generate predicted structures to guide mutational studies.

• Systems biology approaches: Integrate CC_1976 into larger models of C. crescentus cell cycle regulation, similar to the quantitative computational models described by Li et al. :

  • These models can investigate how the three master regulators (DnaA, GcrA, and CtrA) potentially interact with membrane proteins like CC_1976

  • Stochastic simulations can predict cellular responses to perturbations in CC_1976 expression or function

  • In silico testing of hypotheses can prioritize wet-lab experiments

• Translation efficiency prediction: Use computational tools to predict and optimize translation initiation site accessibility, which strongly correlates with protein production levels (Spearman's correlation coefficient Rs = −0.53, P = 3.4 × 10−3) .

This integration of computational and experimental approaches creates a powerful framework for hypothesis generation and testing in CC_1976 research .

What strategies can address low expression yields of recombinant CC_1976?

Low expression yields of membrane proteins like CC_1976 are common challenges. The following methodological interventions can improve yields:

• Translation initiation site optimization: Analyze and modify the 5' region of the coding sequence to ensure optimal accessibility (opening energy ≤12 kcal/mol), which strongly correlates with expression levels .

• Expression strain selection: Test multiple E. coli strains specialized for membrane protein expression (C41(DE3), C43(DE3), Lemo21(DE3)) which contain mutations that reduce toxicity associated with membrane protein overexpression.

• Induction parameters optimization:

  • Reduce induction temperature (16-20°C)

  • Decrease inducer concentration (0.1-0.2 mM IPTG)

  • Extend expression time (overnight or longer)

• Fusion tag approaches: Consider fusion to proteins known to enhance membrane protein expression, such as Mistic, SUMO, or maltose-binding protein.

• Co-expression with chaperones: Co-express with chaperone proteins (GroEL/ES, DnaK/J) to assist proper folding.

Stochastic simulation models predict that optimizing translation initiation accessibility can significantly improve protein production, though there may be a trade-off with cell growth rates .

How can researchers troubleshoot purification issues specific to CC_1976?

Purification troubleshooting for CC_1976 should address common membrane protein challenges:

• Protein aggregation:

  • Ensure adequate detergent concentration (typically 2-3× critical micelle concentration)

  • Screen different detergent types (maltoside series, CHAPS, digitonin)

  • Add glycerol (10-20%) to stabilize the protein

  • Include lipids (e.g., cholesterol, E. coli lipid extract) to stabilize native structure

• Low binding to affinity resin:

  • Verify tag accessibility in the folded protein

  • Try different tag positions (N-terminal vs. C-terminal)

  • Optimize binding conditions (buffer composition, salt concentration, pH)

• Proteolytic degradation:

  • Include protease inhibitor cocktails in all buffers

  • Reduce purification time and maintain low temperature

  • Consider adding stabilizing agents like specific lipids or ligands

• Poor purity after IMAC:

  • Optimize imidazole concentration in wash buffers

  • Add secondary purification steps (ion exchange, size exclusion chromatography)

  • Consider alternative affinity tags (Strep-tag II, FLAG tag)

These strategies should be implemented systematically, documenting outcomes to guide further optimization .