Recombinant Desulfovibrio vulgaris Signal recognition particle receptor FtsY (ftsY)

What expression systems are most suitable for recombinant D. vulgaris FtsY production?

Recombinant D. vulgaris FtsY can be successfully expressed in several prokaryotic systems, with E. coli being the most commonly used host. For optimal expression:

• E. coli BL21(DE3): Offers high yield but may result in inclusion body formation

• E. coli C41/C43: Specialized strains for membrane protein expression, showing improved folding

• Homologous expression: Using D. vulgaris itself as host with plasmids like pMO9075 (stable in D. vulgaris Hildenborough) using spectinomycin (100 μg/ml) as selection marker

Comparative expression levels in different bacterial hosts:

For optimal results, use controlled induction conditions with 0.1-0.5 mM IPTG at reduced temperatures (16-25°C) with extended expression times (12-18 hours) .

How can I optimize the solubility of recombinant D. vulgaris FtsY?

FtsY exists in both membrane-associated and cytosolic forms, making solubility optimization critical:

• Codon optimization: Adjust for D. vulgaris codon bias when expressing in E. coli

• Temperature reduction: Lower induction temperature to 16-20°C

• Detergent supplementation: Add mild detergents (0.1-0.5% CHAPS, DDM, or Triton X-100) during lysis

• Co-expression strategies: Express with SRP components to stabilize the protein

• Fusion tags: N-terminal solubility-enhancing tags such as MBP, SUMO, or thioredoxin

For membrane-bound fractions, adaptation of methods from Shen et al. can improve solubility of membrane-associated proteins . Consider solubilizing membrane fractions with a gradient approach, testing different detergent concentrations before proceeding to purification.

What are the optimal methods for studying the two-step membrane binding mechanism of D. vulgaris FtsY?

The two-step membrane binding mechanism (Dynamic mode followed by Stable mode) can be studied using:

• Single-molecule fluorescence microscopy: Label FtsY with fluorescent probes and monitor association/dissociation events on supported lipid bilayers (SLBs). This technique offers high sensitivity for detecting distinct binding modes .

• Liposome flotation assays: Prepare liposomes with varying lipid compositions to test membrane association under different conditions. Quantify FtsY distribution between membrane and soluble fractions.

• FRET-based assays: Develop assays to monitor conformational changes between Dynamic and Stable modes using strategically placed fluorophores.

• Site-directed mutagenesis: Create mutations in the following key regions:

  • αA1 motif (Dynamic mode interactions)

  • αN1 motif (Stable mode interactions)

  • Analyze binding kinetics of mutants compared to wild type

Experimental setup should include controls comparing wild-type FtsY with engineered variants like FtsY-d14 (reduced Dynamic mode), FtsY-NG (αN1 deletion), and pre-organized FtsY to assess the importance of each binding mode .

How does the GTPase activity of D. vulgaris FtsY differ from other bacterial homologs?

D. vulgaris FtsY, like other bacterial FtsY proteins, exhibits unique GTPase kinetics compared to classical GTPases:

• Nucleotide binding analysis using fluorescence techniques:

  • Measure nucleotide dissociation rates using mant-GTP/GDP fluorescence

  • Expected results: ~10^5 times higher intrinsic guanine nucleotide dissociation rates than Ras proteins

• Comparative GTPase analysis:

• I-box analysis: Examine the role of the I-box insertion (present in all SRP-type GTPases) as an intrinsic exchange factor that dramatically alters GTPase kinetics .

• Lipid-mediated stimulation assays: Test GTPase activity in the presence of PG/PE liposomes to assess the relationship between membrane binding and GTPase activation.

How can I establish a functional in vitro reconstitution system for D. vulgaris FtsY?

A functional reconstitution system requires:

• Purified components:

  • Recombinant D. vulgaris FtsY (with or without tags)

  • D. vulgaris SRP (Ffh + 4.5S RNA or equivalent)

  • Ribosomes (can use E. coli ribosomes as substitute)

  • Lipid vesicles (optimized composition based on D. vulgaris membrane)

• Assembly protocol:

  • Prepare lipid vesicles (70% PE, 20% PG, 10% cardiolipin as starting point)

  • Incorporate SecYEG translocon if studying complete targeting

  • Add ribosomes carrying nascent membrane proteins

  • Add SRP and monitor FtsY recruitment

• Analysis methods:

  • GTPase assays to monitor FtsY activity

  • Fluorescence microscopy for tracking dynamics

  • Membrane flotation to assess targeting efficiency

For cell-free expression approaches, consider PURE systems that allow controlled reconstitution of all components. Adapt protocols from cell-free gene expression systems discussed in source , particularly sections addressing membrane protein expression.

What methods can detect the interaction between D. vulgaris FtsY and the SecY translocon?

Direct interaction between FtsY and the SecY translocon can be assessed by:

• Co-immunoprecipitation: Using antibodies against FtsY to pull down associated SecY components.

• Site-specific crosslinking: Incorporate UV-activatable crosslinkers at key positions in FtsY, particularly in regions implicated in membrane interactions.

• Split fluorescent protein complementation: Fuse complementary fragments of fluorescent proteins to FtsY and SecY to visualize interactions in vivo.

• In vitro binding assays: Purify both components and measure binding constants using:

  • Surface plasmon resonance (SPR)

  • Microscale thermophoresis (MST)

  • Isothermal titration calorimetry (ITC)

• Genomic comparison: Analyze correlation between FtsY and SecY sequence conservation across sulfate-reducing bacteria and related species to identify co-evolving residues .

Why might recombinant D. vulgaris FtsY show different membrane binding properties compared to native protein?

Several factors may explain differences between recombinant and native FtsY behavior:

• Lipid composition effects: D. vulgaris membranes have unique lipid compositions that may not be replicated in heterologous systems. The anaerobic lifestyle of D. vulgaris influences membrane fluidity and composition.

• Post-translational modifications: Potential modifications in native D. vulgaris that are absent in recombinant systems.

• Protein complex formation: Native FtsY exists in complexes with SRP and possibly other factors that stabilize specific conformations.

• Methodology for assessment:

  • Single-molecule approaches may detect subtleties that bulk assays miss

  • Membrane preparation methods can significantly affect observed binding properties

To address these differences, compare recombinant proteins expressed in different systems and incorporate native membrane extracts in binding studies when possible.

How can I address iron incorporation issues when expressing D. vulgaris proteins in E. coli?

D. vulgaris proteins often require proper iron incorporation, as demonstrated with the expression of iron-containing proteins like rubrerythrin :

• Growth media optimization:

  • Supplement with iron sources (ferrous sulfate, 50-100 μM)

  • Use iron-rich media formulations

  • Consider anaerobic culture conditions to maintain iron in reduced state

• In vitro iron incorporation:

  • For iron-deficient expressed protein, dissolve in 3M guanidinium chloride

  • Add Fe(II) anaerobically

  • Dilute the denaturant gradually to allow proper refolding

• Co-expression strategies:

  • Co-express iron-sulfur cluster biogenesis proteins

  • Ensure appropriate cytosolic redox conditions during expression

• Verification methods:

  • UV-vis spectroscopy

  • Mössbauer spectroscopy

  • EPR to confirm proper iron incorporation

While FtsY itself is not an iron-containing protein, these considerations are important when working with other D. vulgaris proteins in the same research program.

How can recombinant D. vulgaris FtsY be used to study differences in protein targeting between aerobic and anaerobic bacteria?

Comparative studies between D. vulgaris FtsY and aerobic bacterial counterparts can reveal adaptations in protein targeting machinery:

• Membrane association comparison:

  • Compare lipid preferences between D. vulgaris FtsY and E. coli FtsY

  • Analyze membrane binding kinetics under aerobic vs. anaerobic conditions

  • Examine the effect of oxidative stress on FtsY function

• Cross-species complementation:

  • Test if D. vulgaris FtsY can complement E. coli FtsY deletion mutants

  • Identify conditions where complementation succeeds or fails

• Specialized targeting substrates:

  • Identify D. vulgaris proteins with unusual signal sequences

  • Test targeting efficiency of these proteins with FtsY from different species

• Evolutionary analysis:

  • Construct phylogenetic trees of FtsY proteins across aerobic/anaerobic species

  • Identify adaptive mutations correlating with anaerobic lifestyle

This research direction could provide insights into how protein targeting systems adapted to different environmental conditions throughout bacterial evolution.

What experimental approaches can be used to study the subcellular localization dynamics of D. vulgaris FtsY?

Advanced imaging approaches to study FtsY localization include:

• Fluorescent protein fusions:

  • Create C-terminal fusions that preserve membrane interactions

  • Use photoactivatable fluorescent proteins for super-resolution imaging

  • Apply techniques similar to those used for studying S. putrefaciens FtsY

• Structured illumination microscopy (SIM):

  • Visualize distribution patterns of FtsY relative to cell membrane

  • Compare with ribosome distribution using L1 protein as marker

• Single-molecule tracking:

  • Apply YFP-bleaching single molecule/particle tracking techniques

  • Use acquisition rates of ~16ms to capture movement of protein complexes

  • Analyze distinct mobility fractions (static, medium-fast, and high mobility)

• Gaussian mixture modeling (GMM):

  • Analyze tracking data to identify distinct subpopulations

  • Compare distributions under different conditions (antibiotic treatment, depletion studies)

  • Create heat maps to visualize diffusive populations

Expected results would show three distinct mobility fractions similar to what has been observed in other bacteria: a static fraction engaged in translation, medium-fast fractions in transition states, and high mobility populations representing freely diffusing molecules.

How might studies of D. vulgaris FtsY contribute to understanding extremophile adaptation mechanisms?

D. vulgaris thrives in anaerobic, often sulfide-rich environments that would be toxic to many organisms. Studies of its FtsY can reveal:

• Membrane adaptations:

  • How protein targeting machinery functions in membranes adapted to extreme conditions

  • Special mechanisms for maintaining protein homeostasis under energy limitation

• Protein stability considerations:

  • Adaptations in FtsY structure that enhance stability under sulfidic conditions

  • Potential protection mechanisms against metal toxicity

• Co-evolution with substrate proteins:

  • How FtsY recognizes and processes specialized membrane proteins unique to sulfate-reducing bacteria

  • Targeting signals specific to proteins involved in anaerobic respiration

• Ecological implications:

  • How efficient membrane protein targeting contributes to D. vulgaris survival in competitive anaerobic niches

  • Energy conservation strategies in protein targeting under energy-limited conditions

These studies would connect protein targeting research with broader questions in microbial ecology and evolution.

What approaches can integrate structural biology and systems biology to study FtsY's role in the D. vulgaris membrane proteome?

Integrative approaches combining structural and systems biology include:

• Structural proteomics:

  • Identify the complete set of membrane proteins dependent on FtsY-mediated targeting

  • Map interaction networks using crosslinking mass spectrometry

• Conditional depletion studies:

  • Create conditional FtsY expression systems in D. vulgaris

  • Analyze proteome-wide changes upon FtsY depletion

  • Identify proteins most sensitive to targeting defects

• Cryo-electron tomography:

  • Visualize FtsY-ribosome-translocon complexes in native membrane environments

  • Compare ultrastructural differences between wild-type and FtsY-depleted cells

• Computational modeling:

  • Develop models integrating protein targeting with cellular energetics

  • Simulate effects of perturbations in the targeting machinery

This integrated approach would provide a systems-level understanding of FtsY's role in the context of D. vulgaris adaptation to its environmental niche.