Recombinant Rhodobacter capsulatus 60 kDa chaperonin (groL), partial
Description
Functional Role in R. capsulatus
GroL in R. capsulatus is a cytoplasmic chaperonin critical for maintaining proteostasis under stress. Proteomic studies identified GroL as a high-abundance protein under standard growth conditions, unaffected by copper availability (unlike other Cu-responsive proteins) . Its roles include:
-
Protein Folding: Assists in refolding denatured proteins via iterative ATP hydrolysis.
-
Stress Adaptation: Stabilizes metabolic enzymes (e.g., malate dehydrogenase, isocitrate dehydrogenase) during oxidative stress .
-
Complex Assembly: Likely collaborates with co-chaperonins (e.g., GroES homologs) for substrate release, analogous to E. coli GroEL-GroES systems .
Recombinant Production and Purification
The partial GroL is produced in E. coli with a His-tag for affinity chromatography. Key steps and parameters:
-
Expression: Induced in E. coli BL21(DE3) or similar strains under IPTG induction.
-
Purification: Nickel-NTA chromatography followed by size-exclusion polishing .
-
Formulation: Stabilized in Tris-HCl (pH 8.0), NaCl, and glycerol to prevent aggregation .
Stability:
-
Short-term: 4°C for 2–4 weeks.
-
Long-term: -20°C with 0.1% HSA/BSA to mitigate freeze-thaw degradation .
Research Applications
While direct studies on recombinant R. capsulatus GroL are sparse, homologous systems suggest utility in:
-
Enzyme Refolding: Assisting in vitro reconstitution of oxygen-labile metalloproteins (e.g., nitrogenase).
-
Stress Response Studies: Probing proteostatic mechanisms under photooxidative or heavy-metal stress.
-
Structural Biology: Serving as a scaffold for crystallizing client proteins.
Comparative Proteomic Insights
A 2020 study profiling R. capsulatus under copper stress noted GroL’s constitutive expression, contrasting with copper-responsive proteins like CutO (multicopper oxidase) and CcoA (copper importer) . This stability highlights its essential housekeeping role.
Key Proteomic Data:
| Condition | GroL Abundance (LFQ Intensity) | Notable Interactors |
|---|---|---|
| Baseline (0.3 μM Cu) | High (~top 5% of proteome) | Ribosomal proteins, EF-Tu, EF-G |
| Cu Excess (5 μM) | No significant change | ATP synthase subunits |
| Cu Depletion (5 mM BCS) | No significant change | GA3P dehydrogenase, trigger factor |
Limitations and Future Directions
Current gaps include:
-
Lack of structural data for R. capsulatus GroL.
-
Underexplored interactions with client proteins in photosynthetic pathways.
-
Engineered variants (e.g., substrate-binding mutants) remain uncharacterized.
