Recombinant Caulobacter crescentus UPF0391 membrane protein CCNA_00709 (CCNA_00709)

What is UPF0391 membrane protein CCNA_00709 and what is its basic structure?

UPF0391 membrane protein CCNA_00709 is a small 60-amino acid membrane protein from the bacterium Caulobacter crescentus. It belongs to the UPF0391 protein family, which consists of uncharacterized proteins with predicted membrane-associated functions. The "UPF" designation (Uncharacterized Protein Family) indicates that while the protein has been identified, its precise biological function remains to be fully elucidated .

The protein has been identified in mass spectrometry studies of bacterial proteomes, particularly through Liquid Extraction Surface Analysis Mass Spectrometry (LESA-MS) . Based on its sequence characteristics and classification, CCNA_00709 is predicted to be an integral membrane protein containing transmembrane domains. For research purposes, the protein has been successfully expressed recombinantly with various tags, including His-tags, to facilitate purification and study .

What is the amino acid sequence of UPF0391 membrane protein CCNA_00709?

The complete amino acid sequence of UPF0391 membrane protein CCNA_00709 consists of 60 residues as follows:

MLKWAIILAIVALIAGALGFSGLAGAAAGVAKILFFLFLVGFVLVLLLGGTVFKAATGPK

Analysis of this sequence reveals characteristic features of membrane proteins, including:

• Multiple hydrophobic stretches that likely form transmembrane domains

• A predominance of aliphatic amino acids (alanine, leucine, isoleucine, valine) typical of membrane-spanning regions

• Glycine residues strategically positioned to provide flexibility in membrane-spanning segments

• Limited charged residues, with those present likely positioned at membrane interfaces or solvent-exposed regions

What approaches are most effective for predicting structural features of CCNA_00709?

For predicting structural features of membrane proteins like CCNA_00709, researchers should employ a multi-faceted approach combining computational methods with experimental validation:

• Transmembrane domain prediction:

  • TMHMM, HMMTOP, or Phobius algorithms to identify potential membrane-spanning regions

  • The hydrophobic stretches in CCNA_00709's sequence suggest multiple transmembrane segments

  • Hydropathy plots to visualize the distribution of hydrophobic regions

• Secondary structure prediction:

  • PSIPRED or JPred for predicting alpha-helical or beta-strand regions

  • Specialized membrane protein secondary structure predictors like MEMSAT

  • Analysis suggests the transmembrane segments likely form alpha-helical structures

• Topology prediction:

  • TOPCONS or CCTOP to predict orientation relative to the membrane

  • SignalP for signal peptide prediction

  • Experimental validation using reporter fusions or selective permeabilization assays

• Homology modeling:

  • HHpred or Phyre2 to identify structural templates

  • SWISS-MODEL or I-TASSER for generating 3D models

  • Molecular dynamics simulations to refine models in a membrane environment

For experimental validation of predictions, methods like protease protection assays, site-directed labeling, and fusion protein approaches provide critical data to complement computational predictions .

What expression systems are available for producing recombinant CCNA_00709?

Several expression systems have been developed for the recombinant production of CCNA_00709, each offering distinct advantages depending on research requirements:

The His-tagged version expressed in E. coli has been well-characterized and is available as a lyophilized powder with a purity greater than 90% as determined by SDS-PAGE . For specialized applications such as protein-protein interaction studies, biotinylated versions using the AviTag-BirA technology are available, allowing for specific in vivo biotinylation and subsequent immobilization or detection .

When selecting an expression system, researchers should consider how their experimental goals might be affected by protein folding, post-translational modifications, and the potential impact of the expression environment on protein structure and function.

What purification strategies yield the highest purity of CCNA_00709?

Achieving high purity of membrane proteins like CCNA_00709 requires specialized approaches to address their hydrophobic nature. The following purification strategy has proven effective:

• Cell lysis and membrane isolation:

  • Mechanical disruption (sonication, French press) followed by differential centrifugation

  • Low-density cell culture conditions improve membrane protein enrichment

  • Careful separation of cytoplasmic and membrane fractions

• Solubilization optimization:

  • Screening of detergents (DDM, LDAO, OG) at various concentrations

  • Detergent solubilization of protein pellets improves specificity for membrane proteins

  • Inclusion of stabilizing agents (glycerol, specific lipids) in solubilization buffers

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged CCNA_00709

  • Carefully optimized binding and washing conditions to minimize non-specific binding

  • Gradual elution with increasing imidazole concentration (typically 20-250 mM)

• Secondary purification steps:

  • Size exclusion chromatography to remove aggregates and further purify protein

  • Ion exchange chromatography if additional purification is needed

  • Removal of the affinity tag if required for downstream applications

The final product has been reported to achieve greater than 90% purity as determined by SDS-PAGE , making it suitable for a wide range of biochemical and structural studies.

How can researchers optimize CCNA_00709 expression in E. coli?

For optimal expression of CCNA_00709 in E. coli, researchers should systematically optimize multiple parameters:

• Strain selection:

  • BL21(DE3) derivatives are commonly used for membrane protein expression

  • C41(DE3) and C43(DE3) strains are engineered specifically for membrane protein expression

  • Lemo21(DE3) allows tunable expression through rhamnose-dependent regulation

• Expression vector design:

  • Incorporation of appropriate fusion tags (His-tag has been successful)

  • Use of tightly controlled promoters (T7lac, araBAD)

  • Codon optimization for E. coli expression

  • Inclusion of ribosome binding site optimization

• Growth and induction conditions:

  • Temperature: Lower temperatures (16-25°C) often improve membrane protein folding

  • Growth media: Rich media for higher biomass vs. minimal media for controlled expression

  • Induction timing: Typically at mid-log phase (OD600 ~0.6-0.8)

  • Inducer concentration: Lower IPTG concentrations (0.1-0.5 mM) often yield better results for membrane proteins

• Environmental variables:

  • Aeration rates affect membrane composition and protein integration

  • Media supplementation with specific lipids or membrane-stabilizing compounds

  • Addition of chemical chaperones (glycerol, betaine, DMSO at low concentrations)

• Harvest timing optimization:

  • Monitoring expression over time to determine optimal harvest point

  • Balancing protein yield with potential toxicity or inclusion body formation

Systematic optimization of these parameters has enabled successful production of CCNA_00709 with high purity (>90%) suitable for a variety of research applications .

What post-translational modifications have been identified in CCNA_00709?

Mass spectrometry studies have revealed important post-translational modifications in CCNA_00709:

• N-terminal formylation:

  • A formylation at the N-terminus has been identified through Liquid Extraction Surface Analysis Mass Spectrometry (LESA-MS)

  • This modification was not previously reported in the UniProt database, representing a novel finding

  • The formylation likely derives from the initiator formyl-methionine, which is typically removed in many bacterial proteins

• Detection methodology:

  • The modification was discovered using LESA-MS coupled with collision-induced dissociation (CID) in the ion trap using helium gas at normalized collision energy of 35%

  • MS/MS spectra were recorded in the Orbitrap at a resolution of 120,000 at 400 m/z

  • Multiple coadded microscans (30 per scan) were used to improve signal quality

• Functional implications:

  • Retention of the formyl group may affect protein stability or protection from aminopeptidases

  • The modification could influence membrane insertion or protein-protein interactions

  • It may serve as a marker for proper protein processing in the bacterial cell

This discovery highlights the importance of comprehensive characterization of post-translational modifications in understanding membrane protein biology and function.

How can mass spectrometry be optimized for studying CCNA_00709?

Optimizing mass spectrometry approaches for membrane proteins like CCNA_00709 requires specialized techniques to address their hydrophobic nature and often low abundance:

• Sample preparation strategies:

  • Detergent-based solubilization followed by detergent removal compatible with MS

  • Gel-assisted digestion methods improve results for membrane proteins

  • Filter-aided sample preparation (FASP) for effective detergent removal

  • Chemical cleavage methods (CNBr, BNPS-skatole) as alternatives to enzymatic digestion

• Digestion optimization:

  • Use of multiple proteases beyond trypsin (chymotrypsin, Glu-C) to improve coverage

  • Extended digestion times (overnight or longer)

  • Addition of organic solvents (10-20% acetonitrile) to improve solubility during digestion

  • Acid-labile detergents that decompose prior to MS analysis

• Instrumentation considerations:

  • Liquid Extraction Surface Analysis Mass Spectrometry (LESA-MS) enables direct protein analysis from bacterial colonies

  • High-resolution MS/MS using Orbitrap analyzers at resolution of 120,000 at 400 m/z

  • Multiple fragmentation techniques (CID, HCD, ETD) provide complementary sequence information

  • Longer LC gradients (90+ minutes) for improved separation of hydrophobic peptides

• Data analysis adjustments:

  • Modified search parameters to account for formylation and other PTMs

  • Consideration of unexpected modifications that may occur during sample processing

  • De novo sequencing approaches for regions with poor coverage

  • Specialized membrane protein databases to improve identification rates

These optimized approaches have successfully identified CCNA_00709 and revealed its N-terminal formylation, demonstrating their effectiveness for membrane protein analysis .

What are recommended approaches for studying protein-protein interactions of CCNA_00709?

Investigating protein-protein interactions involving membrane proteins like CCNA_00709 requires specialized techniques that accommodate their hydrophobic nature:

• In vivo crosslinking approaches:

  • Chemical crosslinking with membrane-permeable reagents (DSP, formaldehyde)

  • Photo-crosslinking with genetically encoded photo-reactive amino acids

  • Proximity-dependent biotin labeling (BioID, TurboID) to identify neighboring proteins

• Affinity-based methods:

  • Pull-down assays using His-tagged CCNA_00709

  • Streptavidin-based pulldowns with biotinylated protein variants

  • Careful detergent selection to maintain native interactions during solubilization

  • Stringent washing protocols to minimize non-specific binding

• Membrane-specific interaction techniques:

  • Bimolecular fluorescence complementation (BiFC) in bacterial systems

  • Fluorescence resonance energy transfer (FRET) for detecting interactions in membranes

  • Single-molecule tracking to detect co-diffusion of protein complexes

  • Liposome/nanodisc reconstitution followed by analytical ultracentrifugation

• Mass spectrometry-based approaches:

  • Crosslinking mass spectrometry (XL-MS) to map interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify binding regions

  • LESA-MS has shown capability for detecting membrane proteins and could be adapted for interaction studies

• Computational prediction and validation:

  • Molecular docking simulations to predict potential interaction partners

  • Coevolution analysis to identify functionally linked proteins

  • Experimental validation of predicted interactions using targeted approaches

Given CCNA_00709's small size (60 amino acids), it likely functions as part of larger protein complexes, making interaction studies particularly important for understanding its biological role.

How can researchers investigate the evolutionary conservation of UPF0391 family proteins?

To comprehensively investigate evolutionary aspects of UPF0391 family proteins like CCNA_00709, researchers should implement a multi-faceted approach:

• Sequence-based comparative analysis:

  • BLAST searches across diverse bacterial genomes to identify homologs

  • Multiple sequence alignment to identify conserved residues and regions

  • Calculation of conservation scores for each amino acid position

  • Phylogenetic tree construction to visualize evolutionary relationships and possible subfamilies

• Structure-based evolutionary assessment:

  • Homology modeling of homologs from different species

  • Comparison of predicted transmembrane topologies across homologs

  • Identification of conserved structural motifs that may be functionally important

  • Mapping of sequence conservation onto structural models

• Genomic context analysis:

  • Examination of gene neighborhoods across species

  • Identification of consistently co-occurring genes

  • Analysis of operon structures in different organisms

  • Detection of potential horizontal gene transfer events

• Experimental cross-species studies:

  • Heterologous expression to test functional conservation

  • Cross-species complementation of knockout phenotypes

  • Domain swapping experiments between distant homologs

  • Comparison of protein-protein interaction networks across species

Studies have already revealed that CCNA_00709 shares 100% sequence homology with a protein from Lelliotia amnigena , while other homologs with varying degrees of similarity exist in other bacterial species. This remarkable conservation suggests strong evolutionary pressure to maintain this protein's sequence, pointing to an important cellular function.

What are the technical challenges in crystallizing membrane proteins like CCNA_00709?

Crystallization of membrane proteins like CCNA_00709 presents several technical challenges that researchers must systematically address:

• Protein extraction and stability issues:

  • Maintaining protein stability outside its native membrane environment

  • Selecting appropriate detergents that maintain native structure

  • Preventing protein aggregation during concentration

  • Achieving sufficient protein quantities (typically milligrams) of homogeneous sample

• Crystallization challenges specific to membrane proteins:

  • Limited polar surface area for crystal contact formation

  • Detergent micelles obscuring potential crystal contacts

  • Phase separation in crystallization drops

  • Conformational heterogeneity leading to poor crystal order

• Size-specific challenges for CCNA_00709:

  • Small size (60 amino acids) provides limited surface for crystal contacts

  • High hydrophobicity-to-size ratio

  • Potential flexibility in membrane-spanning regions

  • Possible requirement for lipid or protein partners for stable structure

• Strategic approaches to overcome these limitations:

  • Fusion protein approaches (T4 lysozyme, BRIL, or antibody fragment fusions)

  • Lipidic cubic phase (LCP) or sponge phase crystallization

  • Antibody fragment co-crystallization to increase polar surface area

  • Screening multiple detergents, additives, and crystallization conditions

  • Consideration of nanobody or synthetic binding protein co-crystallization

• Alternative structural techniques when crystallization proves challenging:

  • NMR spectroscopy for solution or solid-state structural determination

  • Cryo-electron microscopy with lipid nanodiscs

  • Hybrid approaches combining lower-resolution data with computational modeling

These technical challenges explain why structural information for many small membrane proteins remains limited, highlighting the need for innovative approaches to structural determination.

How can CCNA_00709 be studied in native-like membrane environments?

To study CCNA_00709 in conditions that closely resemble its native membrane context, researchers should consider the following methodological approaches:

• Membrane mimetic systems:

  • Reconstitution into liposomes with defined lipid composition

  • Incorporation into nanodiscs with MSP (membrane scaffold protein) belts

  • Amphipol-stabilized protein preparations

  • Native membrane vesicles isolated from Caulobacter crescentus

• In situ structural approaches:

  • Solid-state NMR of membrane-embedded protein

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

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

  • Liquid Extraction Surface Analysis Mass Spectrometry (LESA-MS) for direct analysis from bacterial colonies

• Functional characterization in membranes:

  • Fluorescence-based assays for monitoring conformational changes

  • Surface plasmon resonance with membrane-anchored protein

  • Atomic force microscopy for topological studies

  • Electrophysiological measurements if transport function is suspected

• Cellular localization studies:

  • Fluorescent protein fusions for live-cell imaging

  • Immunogold labeling for electron microscopy

  • Super-resolution microscopy techniques (STORM, PALM)

  • Correlative light and electron microscopy (CLEM)

• Improved extraction methods:

  • Specialized protocols developed for Caulobacter crescentus membrane proteins

  • Use of low-density cell culture conditions for improved membrane protein enrichment

  • Detergent solubilization of protein pellets for enhanced specificity

These approaches provide complementary information about CCNA_00709's structure, dynamics, interactions, and function in environments that maintain the protein's native conformational state and activity.

What are best practices for storing and handling recombinant CCNA_00709?

Proper storage and handling are critical for maintaining the structural integrity and functional properties of recombinant CCNA_00709:

• Initial processing upon receipt:

  • Brief centrifugation of vials before opening to bring contents to the bottom

  • Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Addition of glycerol to a final concentration of 5-50% (with 50% as the recommended default)

• Storage recommendations:

  • Short-term storage: Working aliquots can be stored at 4°C for up to one week

  • Long-term storage: Store at -20°C/-80°C with appropriate cryoprotectants

  • Avoid repeated freeze-thaw cycles which can lead to protein denaturation and aggregation

• Buffer conditions:

  • Standard storage buffer: Tris/PBS-based buffer with 6% Trehalose, pH 8.0

  • Trehalose serves as a stabilizing agent to protect protein structure during freeze-thaw cycles

  • Neutral to slightly alkaline pH (7.5-8.0) helps maintain protein stability

• Handling during experiments:

  • Maintain on ice when thawed for experiments

  • Use low-binding plastic tubes and pipette tips to prevent protein loss

  • Centrifuge briefly before opening tubes to collect condensation

  • Minimize exposure to air/water interfaces which can cause denaturation

• Quality control procedures:

  • Periodic SDS-PAGE analysis to check for degradation

  • Measurement of concentration after extended storage

  • Functional assays to confirm retention of biological activity

Following these guidelines ensures optimal protein quality for experimental use and maximizes the shelf-life of valuable recombinant protein preparations.