Recombinant Zymomonas mobilis UPF0060 membrane protein ZMO1566 (ZMO1566)

What is Zymomonas mobilis UPF0060 membrane protein ZMO1566?

Zymomonas mobilis UPF0060 membrane protein ZMO1566 is a full-length protein (107 amino acids) belonging to the UPF0060 family of membrane proteins. It is encoded by the ZMO1566 gene in Zymomonas mobilis subsp. mobilis. The protein contains membrane-spanning domains and is characterized by its hydrophobic regions that facilitate integration into cellular membranes. As a membrane-associated protein, ZMO1566 likely plays a role in cellular processes such as molecular transport, signaling, or maintaining membrane integrity, although its precise function requires further characterization through experimental studies .

How should recombinant ZMO1566 be stored to maintain stability?

Based on standardized protocols for similar membrane proteins, recombinant ZMO1566 should be stored according to the following guidelines:

Repeated freeze-thaw cycles should be avoided as they can significantly reduce protein stability and activity .

What expression systems are recommended for recombinant ZMO1566 production?

For successful expression of recombinant ZMO1566, E. coli expression systems have been validated and shown to produce functional protein. When selecting an expression system, researchers should consider:

• E. coli system advantages:

  • Cost-effective and rapid expression

  • Well-established protocols for membrane protein expression

  • Compatible with N-terminal His-tag fusion designs

• Potential challenges to address:

  • Codon optimization may be necessary due to differences between Zymomonas mobilis and E. coli codon usage

  • Hydrophobic membrane proteins often require specialized strains designed for membrane protein expression

  • Expression conditions including temperature, induction timing, and media composition need optimization for maximum yield

• Alternative systems:

  • For studies requiring post-translational modifications, yeast or insect cell systems might be considered

  • Cell-free expression systems can be useful for difficult-to-express membrane proteins

What purification strategies yield the highest purity and activity for recombinant ZMO1566?

A multi-step purification strategy is recommended for obtaining high-purity, functional ZMO1566:

• Initial membrane isolation:

  • Lyse cells using mechanical disruption (sonication or French press)

  • Separate membrane fraction by ultracentrifugation (typically 100,000 × g for 1 hour)

  • Solubilize membrane proteins using appropriate detergents (e.g., n-dodecyl-β-D-maltoside or CHAPS)

• Affinity chromatography:

  • Utilize the N-terminal His-tag for IMAC (Immobilized Metal Affinity Chromatography)

  • Include stepwise imidazole concentration increases during elution to separate truncated products from full-length protein

  • Consider using increasing imidazole concentrations (10-250 mM) during washing steps to improve purity

• Secondary purification:

  • Size exclusion chromatography to remove aggregates and ensure monodispersity

  • Ion exchange chromatography for further purification if needed

• Quality control:

  • SDS-PAGE analysis to confirm purity (>90% purity is typically achievable)

  • Western blotting with anti-His antibodies to confirm identity

  • Mass spectrometry to verify the full-length sequence

What computational methods can predict the tertiary structure of ZMO1566?

Several computational approaches can be employed to predict the structure of ZMO1566:

• Homology modeling:

  • Identify structural homologs in the Protein Data Bank

  • Use tools like MODELLER, SWISS-MODEL, or Phyre2

  • Validate models using energy minimization and Ramachandran plot analysis

• Ab initio prediction:

  • AlphaFold2 has revolutionized structure prediction for proteins with limited homologs

  • RoseTTAFold provides alternative modeling approaches

  • I-TASSER can generate hybrid models combining threading and ab initio methods

• Membrane protein-specific tools:

  • MEMSAT for transmembrane topology prediction

  • TMHMM for transmembrane helix prediction

  • PredictProtein for identifying functional motifs within the structure

• Molecular dynamics simulations:

  • Embed predicted structures in virtual membrane environments

  • Assess stability and conformational changes

  • Identify potential functional domains through dynamics analysis

How can researchers experimentally determine the membrane topology of ZMO1566?

Determining the membrane topology of ZMO1566 requires a combination of complementary experimental approaches:

• Cysteine scanning mutagenesis:

  • Systematically replace residues with cysteine throughout the sequence

  • Test accessibility using membrane-impermeable sulfhydryl reagents

  • Accessible regions indicate cytoplasmic or periplasmic exposure

• Fusion protein approaches:

  • Create fusion proteins with reporter enzymes (e.g., alkaline phosphatase, β-galactosidase)

  • Reporter activity indicates cellular localization of the fusion point

  • Generate a series of truncations to map the entire topology

• Protease protection assays:

  • Treat membrane vesicles with proteases

  • Analyze protected fragments by mass spectrometry

  • Compare results with and without membrane permeabilization

• Fluorescence techniques:

  • Site-specific labeling with environment-sensitive fluorophores

  • Measure accessibility and environment polarity

  • Use FRET pairs to determine spatial relationships

How does ZMO1566 potentially interact with other proteins in the bacterial membrane?

Understanding ZMO1566's interactions with other proteins requires multi-faceted experimental approaches:

• Co-immunoprecipitation strategies:

  • Use antibodies against the His-tag to pull down ZMO1566 complexes

  • Perform reverse co-IP with antibodies against suspected partner proteins

  • Analyze results by mass spectrometry to identify interaction partners

• Crosslinking studies:

  • Apply membrane-permeable crosslinking agents

  • Identify crosslinked products by size shifts in SDS-PAGE

  • Utilize mass spectrometry to identify crosslinked residues and interacting proteins

• Protein-protein interaction screens:

  • Bacterial two-hybrid assays adapted for membrane proteins

  • Split-ubiquitin systems for detecting membrane protein interactions

  • Proximity-dependent biotin identification (BioID) for identifying neighboring proteins

• Biophysical confirmation:

  • Surface plasmon resonance to quantify interaction kinetics

  • Microscale thermophoresis for binding affinity measurements

  • Analytical ultracentrifugation to characterize complex formation

How can site-directed mutagenesis be optimized to study ZMO1566 function?

A strategic approach to site-directed mutagenesis should follow these guidelines:

• Target selection criteria:

  • Conserved residues identified through multiple sequence alignments

  • Predicted functional domains or motifs from computational analysis

  • Hydrophilic residues potentially involved in substrate binding

  • Charged residues at predicted membrane interfaces

• Mutation design principles:

  • Conservative mutations: replace with amino acids of similar properties to test specificity

  • Non-conservative mutations: dramatically alter properties to disrupt function

  • Alanine scanning: systematic replacement with alanine to identify essential residues

  • Cysteine substitutions: enable subsequent labeling with thiol-reactive probes

• Validation experiments:

  • Expression level verification by Western blotting

  • Membrane localization confirmation by fractionation

  • Functional assays appropriate to the predicted protein role

  • Thermal stability assessment to detect structural perturbations

How conserved is ZMO1566 across different Zymomonas mobilis strains?

Analysis of ZMO1566 conservation requires:

• Sequence comparison methodology:

  • Collection of ZMO1566 homologs from different Z. mobilis strains

  • Multiple sequence alignment using CLUSTAL Omega or MUSCLE

  • Conservation scoring using Jensen-Shannon divergence or similar metrics

  • Visualization of conservation patterns using tools like ConSurf or WebLogo

• Structural context analysis:

  • Mapping conservation scores onto predicted 3D structures

  • Identifying conserved surface patches that might indicate functional sites

  • Analyzing conservation patterns in transmembrane versus loop regions

• Functional implications:

  • Highly conserved regions often indicate functional importance

  • Variable regions may suggest strain-specific adaptations

  • Correlation between conservation patterns and predicted functional domains

What can phylogenetic analysis reveal about the evolution of ZMO1566?

Phylogenetic analysis of ZMO1566 should include:

• Dataset preparation:

  • Collection of UPF0060 family proteins from diverse bacterial species

  • Inclusion of close homologs from related organisms

  • Multiple sequence alignment with refinement for insertions/deletions

• Tree construction methods:

  • Maximum likelihood methods using tools like RAxML or IQ-TREE

  • Bayesian inference using MrBayes or similar software

  • Selection of appropriate evolutionary models based on likelihood tests

  • Bootstrap analysis to assess node support (typically 1,000 replicates)

• Evolutionary interpretation:

  • Dating divergence events through molecular clock analysis

  • Correlating evolutionary patterns with bacterial ecological niches

  • Identifying potential horizontal gene transfer events

  • Detecting signatures of selection using dN/dS analysis

How can researchers overcome expression challenges with recombinant ZMO1566?

When facing expression difficulties with ZMO1566, consider these approaches:

• Expression optimization strategies:

  • Codon optimization for the expression host

  • Testing multiple promoter systems (T7, tac, arabinose-inducible)

  • Evaluating different E. coli strains (BL21(DE3), C41/C43, Rosetta)

  • Adjusting induction conditions (temperature, IPTG concentration, induction time)

• Fusion partner considerations:

  • Testing solubility-enhancing tags (MBP, SUMO, Trx)

  • Incorporating fusion partners at both N- and C-termini

  • Including protease sites for tag removal

• Toxicity mitigation:

  • Using tight expression control with glucose repression

  • Testing auto-induction media for gradual protein production

  • Employing specialized membrane protein expression strains

• Solubilization approaches:

  • Screening multiple detergents for optimal extraction

  • Testing detergent mixtures for improved solubilization

  • Evaluating novel solubilization agents like styrene maleic acid lipid particles (SMALPs)

What analytical techniques are most effective for verifying proper folding of recombinant ZMO1566?

Assessing the proper folding of ZMO1566 requires multiple complementary techniques:

• Biophysical characterization:

  • Circular dichroism spectroscopy to assess secondary structure content

  • Fluorescence spectroscopy to evaluate tertiary structure integrity

  • Thermal shift assays to determine stability and proper folding

  • Dynamic light scattering to confirm monodispersity

• Functional verification:

  • Ligand binding assays if binding partners are known

  • Activity assays appropriate to predicted function

  • Reconstitution into liposomes to test membrane integration

• Structural integrity assessment:

  • Limited proteolysis to probe for properly folded domains

  • Native PAGE analysis compared to denatured samples

  • Size exclusion chromatography profiles to detect aggregation

  • NMR fingerprinting for structural assessment