Recombinant Ralstonia metallidurans ATP-dependent zinc metalloprotease FtsH (ftsH)

Advanced Research Questions

• What expression systems are optimal for producing functional recombinant R. metallidurans FtsH?

  Several expression systems can be used for producing recombinant R. metallidurans FtsH, each with distinct advantages:

  Expression System	Advantages	Considerations for FtsH Expression
  E. coli BL21(DE3)	High yield, ease of use	May require codon optimization; potential inclusion body formation
  E. coli C41/C43	Specialized for membrane proteins	Better for full-length FtsH with transmembrane domains
  E. coli Rosetta	Supplies rare codons	Helpful if R. metallidurans uses rare codons in ftsH gene
  Cell-free systems	Avoids toxicity issues	Useful for functional domains of FtsH
  Baculovirus/insect cells	Better folding of complex proteins	Higher cost but potentially better activity

  For structural studies, expressing the soluble domain without transmembrane regions in standard E. coli systems with solubility-enhancing tags is recommended. For functional studies, C41/C43 strains with careful induction parameters (lower temperature of 16-18°C, lower IPTG concentration of 0.1-0.5mM) often yield properly folded membrane-associated FtsH .

• What structural domains of FtsH contribute to its metal resistance properties?

  Several structural domains of FtsH likely contribute to metal resistance mechanisms:

  Domain	Position in Sequence	Potential Role in Metal Resistance
  Transmembrane domains	N-terminal region	Membrane anchoring; possibly involved in sensing membrane perturbations caused by metals
  ATPase domain (AAA+)	Central region	Provides energy for conformational changes and substrate processing; may be regulated under metal stress
  Zinc-binding motif (HEXXH)	C-terminal region	Contains catalytic site; zinc binding may be affected by cellular metal homeostasis
  Substrate recognition sites	Various regions	May recognize specific metal-damaged proteins or regulatory factors

  The zinc-binding motif is particularly relevant in the context of metal resistance, as it contains a zinc ion essential for proteolytic activity. In metal-rich environments, competition for metal binding sites or metal-induced conformational changes could potentially alter FtsH activity as part of the cell's adaptive response to metal stress .

• How do ATP binding and hydrolysis affect the conformation and activity of R. metallidurans FtsH?

  ATP binding and hydrolysis induce critical conformational changes in FtsH that are essential for its proteolytic function:

  1. ATP binding to the AAA+ domain induces hexamer formation, the active oligomeric state of FtsH

  2. Upon ATP binding, conformational changes create a central pore for substrate translocation

  3. The ATP-bound state exhibits higher affinity for protein substrates

  4. ATP hydrolysis drives the power stroke that unfolds and translocates substrates into the proteolytic chamber

  5. The energy from ATP hydrolysis enables processing of stable, folded proteins that would otherwise resist degradation

  The ATPase cycle follows this general sequence:
  ATP binding → Conformational change → Substrate binding → ATP hydrolysis → Mechanical unfolding/translocation → Product release → ADP release

  Mutations in the ATP-binding Walker A motif (typically GXXGXGKT/S) abolish both ATPase and protease activities, demonstrating the essential coupling between these functions .

• How can site-directed mutagenesis be used to study functional domains of R. metallidurans FtsH?

  Site-directed mutagenesis provides powerful insights into FtsH structure-function relationships:

  Domain	Target Residues	Expected Effect of Mutation	Research Question Addressed
  ATPase domain	Walker A (K198)	Abolishes ATP binding and hydrolysis	ATP requirement for protease activity
  ATPase domain	Walker B (E269)	Allows ATP binding but prevents hydrolysis	Distinguish binding from hydrolysis effects
  Zinc-binding motif	HEXXH motif (H424, E425, H428)	Eliminates proteolytic activity	Zinc-dependency of protease function
  Transmembrane domain	Hydrophobic residues	Alters membrane association	Role of membrane anchoring in function
  Pore loops	Conserved Tyr residues	Impairs substrate translocation	Substrate processing mechanism
  Substrate-binding regions	Various conserved residues	Alters substrate specificity	Determinants of substrate recognition

  A comprehensive mutagenesis approach would identify conserved residues through sequence alignment with characterized FtsH proteins, generate single amino acid substitutions using PCR-based methods, and compare biochemical properties of mutant proteins with wild-type controls .

• What experimental assays can be used to measure FtsH protease activity?

  Several experimental assays can effectively measure FtsH protease activity:

  Assay Type	Method	Advantages	Limitations
  Fluorogenic substrate assays	Measure cleavage of FITC-labeled casein or custom peptides with fluorescence detection	Quantitative, real-time monitoring, high sensitivity	May not reflect native substrate specificity
  SDS-PAGE-based degradation assays	Incubate FtsH with protein substrate, sample over time, analyze by SDS-PAGE	Visualizes actual substrate degradation, semi-quantitative	Lower throughput, requires larger sample volumes
  Western blot degradation assays	Similar to SDS-PAGE but with immunodetection of specific substrates	Higher sensitivity for specific substrates	Requires antibodies, semi-quantitative
  FRET-based assays	Custom peptides with fluorophore/quencher pairs	Real-time, highly sensitive, adaptable to high-throughput	Requires custom peptide design and synthesis
  In vivo degradation assays	Express substrate-reporter fusions in cells with modulated FtsH levels	Physiologically relevant conditions	Complex system with many variables

  A typical protease assay protocol would include incubation of purified FtsH (0.5-2 μM) with substrate in buffer containing ATP, MgCl₂, and ZnCl₂, with sampling at defined time points (0-60 min). Control reactions should include ATP-omitted samples and heat-inactivated enzyme to confirm ATP-dependency and enzyme-specific activity .

• How can researchers assess the purity and functional integrity of recombinant FtsH preparations?

  Assessment of recombinant FtsH preparations requires multiple complementary approaches:

  For purity assessment:

  • SDS-PAGE with Coomassie or silver staining (expect a band at ~71 kDa for the full-length protein)

  • Western blotting using anti-His antibodies (for His-tagged constructs)

  • Size exclusion chromatography to assess oligomeric state and homogeneity

  • Mass spectrometry for definitive identification and detection of potential contaminants

  For functional integrity assessment:

  • ATPase activity assay measuring ATP hydrolysis rates (colorimetric phosphate detection)

  • Proteolytic activity using fluorogenic model substrates (e.g., FITC-casein)

  • Specific substrate degradation assays using known FtsH substrates

  • Circular dichroism to confirm proper protein folding

  • Thermal shift assays to assess protein stability

  Active preparations should show both ATP hydrolysis and ATP-dependent proteolytic activity, with specific activity values comparable to those reported in the literature for bacterial FtsH proteins .

• What role does FtsH play in stress response pathways in R. metallidurans?

  In R. metallidurans, FtsH likely serves as a central regulator of multiple stress response pathways:

  1. Heat shock response: FtsH typically degrades the heat shock sigma factor σ32 (RpoH) during normal growth, but this degradation is reduced under stress conditions, allowing accumulation of σ32 and induction of heat shock genes.

  2. Membrane stress: FtsH removes misfolded membrane proteins and regulates the levels of certain lipid biosynthesis enzymes, maintaining membrane integrity during stress.

  3. Metal stress response: In metal-resistant R. metallidurans, FtsH may regulate specific proteins involved in metal detoxification or efflux systems, though specific targets remain to be identified.

  4. Oxidative stress: Metal exposure often induces oxidative stress; FtsH may degrade oxidatively damaged proteins and regulate redox-responsive transcription factors.

  The specific proteins targeted by R. metallidurans FtsH under metal stress conditions represent an important area for future research, particularly given this organism's remarkable adaptation to metal-contaminated environments .

• How can researchers design experiments to study the in vivo function of FtsH in R. metallidurans?

  Designing experiments to study in vivo FtsH function in R. metallidurans requires strategic approaches:

  Experimental Approach	Methodology	Information Gained	Technical Considerations
  Conditional knockdown	Inducible antisense RNA or CRISPR interference	Effects of FtsH depletion on viability and stress resistance	Complete knockout may be lethal; requires optimized genetic tools
  Point mutations	Chromosomal integration of catalytically inactive variants	Separate roles of different FtsH domains	Needs efficient site-directed mutagenesis system for R. metallidurans
  Transcriptomics	RNA-seq comparing wild-type and FtsH-depleted strains	Global effects on gene expression during metal stress	Requires careful experimental design to capture direct vs. indirect effects
  Proteomics	MS-based identification of differentially abundant proteins	Identification of potential FtsH substrates	Should include membrane protein enrichment steps
  Substrate trapping	Expression of catalytically inactive FtsH followed by co-IP and MS	Direct identification of FtsH interaction partners	May require optimized immunoprecipitation protocols
  Metal resistance assays	Growth in presence of various metals with normal or depleted FtsH	Metal-specific roles of FtsH	Should test multiple metals and concentrations

  Since R. metallidurans is adapted to metal-rich environments, particular attention should be paid to examining FtsH function under exposure to various heavy metals at different concentrations and combinations .

• What substrate specificity does R. metallidurans FtsH exhibit compared to other bacterial FtsH proteins?

  The substrate specificity of R. metallidurans FtsH has not been comprehensively characterized, but predictions can be made based on conservation patterns and the specialized niche of this organism:

  Common FtsH substrates likely recognized by R. metallidurans FtsH:

  • Heat shock sigma factor σ32 (regulation of heat shock response)

  • Uncomplexed subunits of membrane protein complexes

  • Misfolded membrane proteins

  • Specific short-lived regulatory proteins

  Potential specialized substrates in R. metallidurans:

  • Regulators of metal resistance operons

  • Metal-damaged membrane proteins

  • Components of metal transport systems requiring strict regulation

  • Proteins involved in the cellular response to oxidative stress induced by metals

  Comparative sequence analysis of the substrate-binding regions between R. metallidurans FtsH and other bacterial FtsH proteins might reveal adaptations that reflect its substrate preferences in metal-rich environments .

• How can researchers develop assays to study FtsH's role in metal resistance mechanisms?

  Developing assays to study FtsH's role in metal resistance mechanisms requires multilevel approaches:

  Cellular assays:

  1. Metal tolerance comparison: Compare growth of wild-type vs. FtsH-depleted strains in media containing increasing concentrations of various heavy metals (Cd, Zn, Cu, Co, Ni, etc.)

  2. Metal accumulation: Measure intracellular and membrane-bound metal content using ICP-MS in normal vs. FtsH-depleted cells

  3. Metal efflux kinetics: Track rate of metal export in cells with normal or altered FtsH levels

  4. Membrane integrity: Assess membrane permeability changes during metal exposure with and without functional FtsH

  Molecular assays:

  1. Expression analysis: Monitor expression of known metal resistance genes (from pMOL28, pMOL30, and chromosomal loci) in the presence/absence of FtsH

  2. Protein stability: Measure half-lives of metal resistance proteins in cells with normal or depleted FtsH

  3. Targeted degradation assays: Test if FtsH directly degrades specific metal resistance regulators in vitro

  4. Protein interaction networks: Identify FtsH interaction partners under metal stress using co-immunoprecipitation or crosslinking approaches

  A systematic approach would start with phenotypic characterization of metal sensitivity in FtsH-modified strains, followed by identifying specific metal resistance pathways affected by FtsH alteration .

Methodological Questions

• What analytical techniques are most effective for studying FtsH-substrate interactions?

  Multiple complementary analytical techniques can effectively characterize FtsH-substrate interactions:

  Technique	Application	Advantages	Limitations
  Surface Plasmon Resonance (SPR)	Real-time binding kinetics	Quantitative, label-free, measures kon and koff	Requires immobilization which may affect function
  Isothermal Titration Calorimetry (ITC)	Thermodynamics of binding	Label-free, provides complete thermodynamic profile	Requires significant amounts of purified proteins
  Microscale Thermophoresis (MST)	Binding affinity in solution	Low sample consumption, works in native buffers	Requires fluorescent labeling
  Hydrogen-Deuterium Exchange MS	Mapping interaction interfaces	Identifies specific binding regions, works with large complexes	Complex data analysis, specialized equipment
  Crosslinking coupled with MS	Capturing transient interactions	Can trap short-lived complexes, works in native settings	May capture non-specific interactions
  Cryo-EM	Structural characterization of complexes	Visualizes substrate bound to FtsH complex	Technically challenging, requires specialized equipment

  For R. metallidurans FtsH specifically, these approaches could focus on testing interactions with known metal resistance proteins and regulatory factors to elucidate its role in the metal resistance phenotype .

• How do reconstitution and storage conditions affect the activity of recombinant FtsH?

  Reconstitution and storage conditions significantly impact recombinant FtsH activity:

  Reconstitution considerations:

  • Buffer composition: Typically Tris/PBS-based buffer at pH 8.0

  • Additives needed: 6% Trehalose helps maintain protein stability

  • Concentration: Optimal reconstitution at 0.1-1.0 mg/mL

  • Glycerol addition: 5-50% final concentration recommended for long-term storage

  Storage considerations:

  • Temperature: Store at -20°C/-80°C for long-term storage; working aliquots at 4°C for up to one week

  • Aliquoting: Essential to avoid repeated freeze-thaw cycles

  • Stability concerns: Repeated freezing and thawing significantly reduces activity

  • Activity testing: Regular activity assays should be performed to confirm protein functionality

  The reconstitution procedure should involve brief centrifugation of the vial prior to opening, followed by addition of deionized sterile water to achieve the desired concentration, with glycerol addition for stability .

• What are the key considerations for comparing FtsH from R. metallidurans with FtsH from other bacterial species?

  When comparing FtsH from R. metallidurans with FtsH from other bacteria, researchers should consider:

  Sequence-based considerations:

  • Phylogenetic relationships to determine evolutionary context

  • Conservation of catalytic residues across species

  • Unique sequence features in R. metallidurans FtsH that may relate to metal resistance

  • Comparison with FtsH from other metal-resistant organisms vs. non-resistant species

  Functional considerations:

  • Substrate specificity differences

  • Metal ion requirements and sensitivities

  • ATPase activity under various conditions

  • Response to environmental stressors, particularly metals

  Structural considerations:

  • Differences in membrane-spanning domains

  • Variations in oligomeric assembly

  • Substrate-binding pocket architecture

  A particularly valuable comparison would be between R. metallidurans FtsH and FtsH from Ralstonia solanacearum, as both species contain metal resistance genes but inhabit different ecological niches .