Recombinant Salinibacter ruber ATP-dependent zinc metalloprotease FtsH 2 (ftsH2)

What cellular functions does FtsH2 likely perform in Salinibacter ruber?

As an ATP-dependent zinc metalloprotease, FtsH2 in S. ruber likely plays critical roles in:

• Protein quality control - degrading misfolded or damaged membrane proteins

• Homeostatic balance maintenance - particularly important in extreme halophilic environments

• Regulation of membrane protein composition

• Possible involvement in balancing charge perturbations associated with xanthorhodopsin and ATP synthase activities

The protein may be particularly important in S. ruber due to its need to maintain tight regulation of ion trafficking across membranes in hypersaline environments. Similar to other FtsH proteases, it likely participates in stress response pathways and may contribute to the organism's ability to thrive in extreme conditions .

How does S. ruber FtsH2 relate to other FtsH family proteases?

While the search results don't provide direct comparative information specific to S. ruber FtsH2, studies of other FtsH proteases (particularly in plants) show that these proteins typically:

• Form hexameric structures with buried surface areas that contribute to stability

• May form either homomeric (same subunit) or heteromeric (different subunits) complexes

• Display ATP-dependent proteolytic activity targeted at specific substrates

Based on modeling studies of similar FtsH proteins, the specific arrangement of subunits significantly impacts the stability of the complex. For instance, in plants, heteromeric complexes of FtsH2 and FtsH5 showed increased buried surface area compared to homomeric complexes, suggesting greater thermodynamic stability .

What expression systems are recommended for producing recombinant S. ruber FtsH2?

The commercially available recombinant S. ruber FtsH2 is expressed in E. coli with an N-terminal His-tag . This bacterial expression system appears suitable for producing functional protein. When establishing an expression protocol for S. ruber FtsH2, researchers should consider:

• Expression vector selection: Vectors with strong, inducible promoters (such as T7) are typically suitable for membrane-associated proteins like FtsH2.

• Strain optimization: BL21(DE3) or similar strains are commonly used for expressing proteases, with potential modifications to address toxicity issues.

• Induction conditions: Optimization of temperature, inducer concentration, and duration is critical - lower temperatures (16-25°C) often produce more properly folded membrane proteins.

• Cell lysis and extraction: Careful selection of detergents is essential for extraction of membrane-associated proteins like FtsH2.

The specific methodology used for the commercially available product involves expression with an N-terminal His-tag, which facilitates purification while maintaining protein functionality .

What are the optimal storage conditions for maintaining FtsH2 activity?

According to the product information, purified recombinant S. ruber FtsH2 should be stored as follows:

• Store at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

• For reconstitution, use deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) for long-term storage

• Storage buffer consists of Tris/PBS-based buffer with 6% Trehalose at pH 8.0

These conditions help maintain protein stability and enzymatic activity. The recommendation against repeated freeze-thaw cycles is particularly important as membrane proteins like FtsH2 are often sensitive to denaturation during this process .

How can researchers assess the enzymatic activity of recombinant S. ruber FtsH2?

While the search results don't provide specific assay protocols for S. ruber FtsH2, researchers can adapt standard approaches for ATP-dependent proteases:

• ATP hydrolysis assay: Measuring ATPase activity using colorimetric methods (malachite green) or radiometric assays with γ-³²P-ATP.

• Proteolytic activity assays:

  • Using fluorogenic peptide substrates with cleavage sites recognized by FtsH proteases

  • Monitoring degradation of model substrates using SDS-PAGE or western blotting

  • FRET-based assays using dual-labeled peptides

• Substrate specificity determination:

  • Incubating purified FtsH2 with candidate substrate proteins under varying conditions

  • Mass spectrometry analysis to identify cleavage sites

Researchers should ensure that assay conditions include appropriate zinc concentrations (as it's a zinc metalloprotease) and ATP (as it's ATP-dependent) . Additionally, since S. ruber is a halophile, the effects of salt concentration on enzymatic activity should be systematically evaluated.

What structural techniques are most informative for studying S. ruber FtsH2?

Based on approaches used with similar proteins, the following structural characterization methods would be most valuable:

• X-ray crystallography: While challenging for membrane proteins, this approach can provide atomic-level resolution of protein structure.

• Cryo-electron microscopy (Cryo-EM): Particularly suitable for large complexes like the hexameric FtsH2, as demonstrated with other FtsH proteins .

• Computational modeling: Homology modeling based on related FtsH structures, as demonstrated with plant FtsH proteins . The structural model of the T. thermophilus FtsH (PDB entry 2DHR) has been used as a template for modeling other FtsH proteins using software like GENO3D .

• Mass spectrometry: For analyzing subunit composition and interactions in native complexes.

A combined approach using multiple techniques would provide the most comprehensive structural understanding. For computational modeling, tools like PyMol, Rasmol, and CCP4 have been used to analyze FtsH structures and calculate parameters such as solvent accessibility surface area .

What role might FtsH2 play in the halophilic adaptation of S. ruber?

S. ruber thrives in extremely saline environments, and FtsH2 likely contributes to this adaptation through several mechanisms:

• Membrane protein quality control: Maintaining functional membrane proteins is critical in extreme environments, and FtsH2 likely removes damaged or misfolded proteins from the membrane.

• Ion homeostasis support: S. ruber has complex inward/outward trafficking of ions to maintain internal osmotic balance. FtsH2 may regulate membrane transporters involved in this process .

• Stress response regulation: FtsH2 may degrade regulatory proteins involved in stress response pathways, thus helping the organism adapt to changing environmental conditions.

• Xanthorhodopsin system interaction: S. ruber contains xanthorhodopsin (XR), a light-harvesting/proton-pumping complex present in high amounts in the cell envelope. FtsH2 may be involved in balancing the charge perturbation associated with XR activity .

The importance of homeostatic regulation in halophiles makes proteases like FtsH2 particularly crucial, as they need complementary systems to balance perturbations associated with their specialized membrane proteins .

How does FtsH2 potentially interact with other membrane systems in S. ruber?

While direct experimental evidence from the search results is limited, the following potential interactions can be inferred:

• Xanthorhodopsin (XR) system: The XR complex is present in high amounts in the S. ruber cell envelope and contributes to its energy metabolism. FtsH2 may help balance charge perturbations associated with XR activity .

• Porin regulation: S. ruber, like other bacteria, contains porins that regulate cell trafficking with the environment. FtsH2 might regulate the turnover of these porins, helping maintain cell homeostasis .

• ATP synthase interaction: As suggested for XR, FtsH2 may also play a role in regulating or balancing activities associated with ATP synthase function .

These potential interactions highlight the integrated nature of membrane protein quality control systems in maintaining cellular homeostasis, particularly in organisms living in extreme environments.

How can comparative studies between S. ruber FtsH2 and other FtsH proteases advance our understanding of proteolytic mechanisms?

Comparative studies between S. ruber FtsH2 and other FtsH proteases can yield valuable insights into:

• Adaptation mechanisms to extreme environments: By comparing the structure and function of FtsH2 from halophilic S. ruber with non-halophilic counterparts, researchers can identify specific adaptations that enable function in high-salt conditions.

• Substrate specificity determinants: Comparing the substrate binding regions across different FtsH proteases can reveal how these enzymes achieve specificity.

• Evolutionary conservation and divergence: Analysis of sequence and structural conservation across FtsH family members can highlight essential functional domains versus species-specific adaptations.

Such comparative approaches have been productive in plant systems, where studies of FtsH2 and FtsH5 revealed that heteromeric complexes have greater thermodynamic stability than homomeric ones . Similar principles could be explored with S. ruber FtsH2.

What experimental approaches can resolve contradictory data about FtsH2 function?

When faced with contradictory data regarding FtsH2 function, researchers should consider:

• In vivo vs. in vitro discrepancies: Creating tagged versions of FtsH2 for in vivo studies (similar to the FtsH2-HA approach used in plants) to compare with in vitro biochemical data.

• Genetic approaches:

  • Creating knockout/knockdown strains to assess phenotypic effects

  • Complementation studies with mutant variants to identify critical residues

  • Suppressor screens to identify functional interactions

• Structural biology combined with site-directed mutagenesis: Identifying critical residues through structural analysis and testing their importance through mutagenesis.

• Proteomics approaches: Using techniques like co-immunoprecipitation followed by mass spectrometry to identify interaction partners and substrates in different conditions.

Plant FtsH research has successfully used epitope-tagged versions (e.g., HA-tagged FtsH2) to purify complexes and identify interacting partners, confirming the heteromeric nature of the complexes . Similar approaches could be applied to S. ruber FtsH2.

How might FtsH2 function be affected by environmental stress conditions relevant to S. ruber's natural habitat?

S. ruber inhabits extreme hypersaline environments, and understanding how FtsH2 functions under various stress conditions is critical:

• Salt concentration effects: Systematic analysis of how varying salt concentrations affect:

  • FtsH2 expression levels

  • Complex assembly and stability

  • Substrate recognition and proteolytic activity

• Temperature stress: Investigating how temperature fluctuations, common in hypersaline environments, impact FtsH2 activity.

• Oxidative stress responses: Similar to the role of FtsH2 in plants in responding to singlet oxygen , S. ruber FtsH2 might play roles in oxidative stress responses.

• Nutrient limitation: Examining how FtsH2 activity changes during nutrient limitation, potentially contributing to protein recycling.

Research methodologies should include:

• qRT-PCR for transcript level analysis (similar to methods used in plant studies)

• Proteomics approaches to assess protein abundance changes

• Activity assays under varying conditions

• In vivo phenotypic studies of stress responses in wild-type versus FtsH2-deficient strains