Recombinant Caulobacter crescentus UPF0060 membrane protein CCNA_02055 (CCNA_02055)

What is the biological function of UPF0060 membrane protein CCNA_02055 in Caulobacter crescentus?

While specific functions of CCNA_02055 remain under investigation, comparative analysis with other characterized Caulobacter membrane proteins suggests potential roles in membrane transport, signaling, or maintenance of cell envelope integrity. Based on characterized proteins in the same organism, CCNA_02055 may function in mechanisms similar to RsaFa and RsaFb, which contribute to protein secretion and cellular fitness .

Recommended experimental approaches for function determination:

• Gene deletion studies with phenotypic characterization

• Protein-protein interaction mapping using crosslinking or co-immunoprecipitation

• Complementation studies with related membrane proteins

• Transcriptomic analysis comparing wild-type and deletion mutants

What expression systems are optimal for recombinant production of CCNA_02055?

Based on protocols established for other Caulobacter membrane proteins, the following expression parameters are recommended:

For Caulobacter membrane proteins, expression levels are typically verified using Western blot analysis with antibodies against the affinity tag, with expected yields of 0.1-1 mg purified protein per liter of culture .

What purification strategy yields highest quality CCNA_02055 for structural studies?

A multi-stage purification protocol is recommended:

• Membrane fraction isolation:

  • Cell disruption by sonication or French press

  • Differential centrifugation (10,000×g to remove debris, 100,000×g to collect membranes)

  • Membrane solubilization using n-dodecyl-β-D-maltoside (DDM) at 1% w/v

• Chromatography sequence:

  • Immobilized metal affinity chromatography (IMAC) with imidazole gradient

  • Size exclusion chromatography using Superdex 200

  • Optional ion exchange chromatography for additional purity

• Critical quality control tests:

  • SDS-PAGE with silver staining (>95% purity)

  • Dynamic light scattering (monodispersity assessment)

  • Circular dichroism (secondary structure verification)

  • Thermal stability assay with differential scanning fluorimetry

Similar approaches have been used successfully for other Caulobacter membrane proteins in structural biology applications .

How does CCNA_02055 compare structurally and functionally to characterized Caulobacter crescentus membrane proteins?

Based on analysis of characterized Caulobacter crescentus membrane proteins:

Experimental approaches to establish functional relationships:

• Comparative proteomics in deletion backgrounds

• Cross-complementation studies

• Protein-protein interaction network mapping

• Differential phenotypic analysis under stress conditions

What methodological challenges exist in CCNA_02055 experimental characterization?

Common challenges in Caulobacter membrane protein research and their solutions:

• Protein instability during purification:

  • Screen multiple detergents (DDM, DM, LMNG)

  • Include stabilizing additives (glycerol, specific lipids)

  • Optimize buffer composition (pH 7.0-8.0, 150-300 mM NaCl)

  • Utilize nanodiscs or amphipols for increased stability

• Functional assay limitations:

  • Develop proteoliposome reconstitution systems

  • Establish fluorescence-based binding assays

  • Implement electrophysiological measurements for transport functions

  • Create genetic reporter systems for in vivo activity

• Structural analysis difficulties:

  • Lipidic cubic phase crystallization for X-ray studies

  • Cryo-EM single-particle analysis

  • Solid-state NMR for membrane-embedded regions

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

These approaches have successfully addressed similar challenges with other Caulobacter membrane proteins .

How can protein-protein interactions of CCNA_02055 be systematically identified?

Based on successful interaction studies with Caulobacter membrane proteins:

Important controls and validation steps:

• Reciprocal pull-downs with identified partners

• Confirmation with multiple methodologies

• Functional validation of interactions

• Comparison with known interactomes of related proteins

For Caulobacter membrane proteins like RsaFa and RsaFb, these approaches have successfully identified functional interaction partners in secretion pathways .

What protocols ensure reproducible functional assays for CCNA_02055?

Critical parameters for reproducible membrane protein functional studies:

• Sample preparation standardization:

  • Defined growth medium composition (PYE medium: 0.2% peptone, 0.1% yeast extract, 1 mM MgSO₄, 0.5 mM CaCl₂)

  • Standardized cell harvesting at precise growth phase (OD₆₀₀ = 0.3-0.4 for exponential phase)

  • Consistent membrane isolation procedures

  • Protein quantification using multiple methods (BCA, UV absorption)

• Assay conditions documentation:

  • Temperature control (±0.5°C)

  • pH stability verification

  • Detergent concentration above critical micelle concentration

  • Defined lipid composition for reconstitution

• Quality control metrics:

  • Minimum acceptable protein purity (>90% by SDS-PAGE)

  • Activity thresholds for positive controls

  • Signal-to-noise ratio requirements

  • Technical and biological replicate consistency

Similar quality control standards were essential for functional characterization of RsaFa and RsaFb in S-layer secretion studies .

What approaches can determine if CCNA_02055 contributes to antibiotic resistance?

Building on methodologies used for other Caulobacter membrane proteins:

• Minimum inhibitory concentration (MIC) determination:

  • Compare wild-type and CCNA_02055 deletion strains

  • Use 1.5-fold serial dilutions of antimicrobials in PYE medium

  • Inoculate with mid-exponential phase cultures (1×10⁹ cells/ml)

  • Measure growth inhibition after 18 hours (OD₆₀₀ threshold of 0.137)

• Antimicrobial agents to test:

  • Cell wall synthesis inhibitors (bacitracin)

  • Protein synthesis inhibitors (tetracycline, erythromycin)

  • DNA synthesis inhibitors (novobiocin)

  • Membrane-disrupting agents (SDS, Triton X-100)

  • Heavy metals (uranyl nitrate, zinc chloride)

• Functional validation experiments:

  • Complementation with wild-type CCNA_02055

  • Nitrocefin hydrolysis assay to measure permeability

  • Polymyxin B nonapeptide (PMBN) sensitization test

  • Fluorescent dye accumulation assays

This approach successfully identified antimicrobial resistance functions for RsaFa and RsaFb in Caulobacter crescentus .

How can transcriptomic analysis reveal CCNA_02055 function in cellular pathways?

RNA-sequencing methodology for membrane protein functional analysis:

• Experimental design considerations:

  • Compare wild-type, CCNA_02055 deletion, and complemented strains

  • Harvest cells at early exponential phase (OD₆₀₀ = 0.27)

  • Immediately stabilize RNA (ice-cold ethanol with 5% water-saturated phenol)

  • Include biological triplicates for statistical power

• RNA isolation and quality control:

  • Extract using hot phenol method

  • DNase treatment to remove genomic DNA

  • Verify RNA integrity (A₂₆₀/A₂₈₀ ≥ 2.0)

  • Confirm absence of genomic DNA contamination by PCR

• Data analysis workflow:

  • Map reads to Caulobacter crescentus genome

  • Calculate differential expression (log₂ fold change, adjusted p-value)

  • Perform gene ontology enrichment analysis

  • Validate key findings with RT-qPCR

• Biological interpretation strategies:

  • Comparison with known regulons

  • Identification of co-regulated gene clusters

  • Pathway analysis for metabolic and signaling networks

  • Integration with protein-protein interaction data

This approach successfully characterized transcriptional changes in RsaF mutants, revealing compensatory mechanisms in S-layer export .

What structural features might determine CCNA_02055 membrane integration and topology?

Predicted structural characteristics based on membrane protein analysis:

• Transmembrane domain prediction:

  • Hydropathy analysis suggests multiple membrane-spanning regions

  • Topology models should be validated experimentally using cysteine accessibility scanning

  • PhoA/LacZ fusion analysis can confirm orientation

• Structural motifs of interest:

  • Potential β-barrel structure (common in outer membrane proteins)

  • Alpha-helical bundles (common in inner membrane proteins)

  • Signal sequences and membrane anchoring domains

  • Conserved charged residues in transmembrane regions

• Experimental topology validation:

  • Site-directed cysteine labeling with membrane-permeable and impermeable reagents

  • Protease protection assays with inverted membrane vesicles

  • Epitope insertion at predicted loops with antibody accessibility testing

Similar structural analysis approaches were used to determine the topology of RsaFa and RsaFb, revealing their β-barrel structure consistent with outer membrane localization .

How might post-translational modifications affect CCNA_02055 function?

Potential post-translational modifications and their functional implications:

• Common bacterial membrane protein modifications:

  • Phosphorylation of cytoplasmic domains (signaling)

  • Lipidation (membrane anchoring)

  • Disulfide bond formation (structural stability)

  • Proteolytic processing (activation or regulation)

• Detection methodologies:

  • Mass spectrometry with enrichment strategies

  • Phospho-specific antibodies

  • Mobility shift assays

  • Chemical labeling of modified residues

• Functional impact assessment:

  • Site-directed mutagenesis of modified residues

  • Activity assays comparing modified and unmodified forms

  • Temporal correlation of modifications with cellular events

  • Comparison with modification patterns of homologous proteins

Post-translational regulation may be particularly relevant for membrane proteins involved in stress responses, similar to the antimicrobial resistance functions observed for RsaFa and RsaFb in Caulobacter .

What biophysical methods are most informative for studying CCNA_02055 dynamics?

Advanced biophysical approaches for membrane protein characterization:

• Spectroscopic techniques:

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

  • Fluorescence resonance energy transfer (FRET) using strategically placed fluorophores

  • Nuclear magnetic resonance (NMR) for dynamics of specific domains

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

• Single-molecule approaches:

  • Atomic force microscopy for topography and mechanical properties

  • Single-molecule FRET for conformational dynamics

  • Optical tweezers for measuring interaction forces

  • High-speed atomic force microscopy for real-time dynamics

• Computational integration:

  • Molecular dynamics simulations in explicit membrane environments

  • Normal mode analysis for identification of functionally relevant motions

  • Markov state modeling of conformational transitions

  • Integration of experimental constraints with computational models

These methods have proven valuable for understanding the dynamics of bacterial membrane proteins similar to the GTP-binding protein CgtA, which displays unusual nucleotide exchange properties in Caulobacter .

How can cryo-electron microscopy advance structural understanding of CCNA_02055?

Cryo-EM workflow for membrane protein structural determination:

• Sample preparation optimization:

  • Detergent screening for monodispersity

  • Reconstitution into nanodiscs or amphipols

  • Gradient fixation (GraFix) for stability

  • Addition of binding partners for conformational stabilization

• Data collection parameters:

  • High-end microscope (300 kV) with direct electron detector

  • Motion correction and dose fractionation

  • Defocus range optimization for contrast

  • Collection of 3000-5000 micrographs for sufficient particles

• Data processing workflow:

  • Particle picking (manual and automated)

  • 2D classification for quality assessment

  • Ab initio 3D model generation

  • Refinement with CTF correction and particle polishing

• Validation and interpretation:

  • Resolution assessment by gold-standard FSC

  • Model building with reference to homologous structures

  • Validation using independent datasets

  • Functional interpretation through mapping of conserved residues

Single-particle cryo-EM has revolutionized membrane protein structural biology and would be applicable to CCNA_02055, especially if it forms multi-protein complexes similar to the S-layer export machinery involving RsaFa and RsaFb .

What comparative genomics approaches can contextualize CCNA_02055 research?

Strategic genomic analysis to guide functional studies:

• Phylogenetic distribution analysis:

  • Survey UPF0060 family proteins across bacterial species

  • Identify co-evolution with other membrane components

  • Determine conservation patterns in alpha-proteobacteria

  • Correlate with ecological niches and bacterial lifestyles

• Genomic context examination:

  • Analyze gene neighborhoods across species

  • Identify conserved operonic structures

  • Detect horizontal gene transfer events

  • Map regulatory elements in promoter regions

• Evolutionary rate analysis:

  • Calculate selection pressure on different protein domains

  • Identify rapidly evolving regions (potential specificity determinants)

  • Compare with evolutionary rates of functionally related proteins

  • Detect signatures of adaptive evolution

These approaches can reveal whether CCNA_02055 serves conserved core functions or specialized roles in Caulobacter, similar to the analysis that revealed the specialized roles of RsaFa and RsaFb in S-layer export .

How might CCNA_02055 research contribute to broader understanding of bacterial membrane biology?

Potential broader impacts of CCNA_02055 research:

• Fundamental membrane biology insights:

  • Membrane protein quality control mechanisms

  • Protein-lipid interactions in bacterial membranes

  • Organization of multi-protein complexes in membranes

  • Membrane adaptation to environmental stresses

• Biotechnology applications:

  • Development of novel protein secretion systems

  • Engineering of membrane protein expression hosts

  • Creation of biosensors based on membrane protein functions

  • Design of antimicrobial strategies targeting essential membrane processes

• Comparative systems biology:

  • Integration with whole-cell models of bacterial physiology

  • Cross-species comparison of membrane proteome function

  • Evolutionary trajectories of membrane protein families

  • Principles of membrane protein structure-function relationships

The unique characteristics of Caulobacter crescentus, including its dimorphic life cycle and asymmetric division, provide valuable context for membrane protein research that complements studies in other model bacterial systems .