Related Experiment Videos
Unfolding thermodynamics of the tetrameric chaperone, SecB
V G Panse1, C P Swaminathan, J J Aloor
1Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Biochemistry
|March 1, 2000
Summary
SecB, a chaperone protein in E. coli, is a stable tetramer that unfolds reversibly into monomers. Its stability decreases with increasing pH, and substrate binding likely occurs at a surface site.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Folding
Background:
- SecB is a cytosolic tetrameric chaperone in Escherichia coli.
- It maintains polypeptides in a translocation-competent state for export.
Purpose of the Study:
- To investigate the thermodynamics of SecB unfolding.
- To understand the structural stability and substrate binding characteristics of SecB.
Main Methods:
- High-sensitivity differential scanning calorimetry (DSC).
- Spectroscopic methods.
- Denaturant-mediated unfolding studies.
- Free thiol reactivity assays.
Main Results:
- Thermal unfolding is reversible, following a two-state transition from tetramer to monomers.
- Increasing pH significantly decreases tetramer stability (Tm decreases from 341.3 K at pH 6.5 to 332.6 K at pH 9.5).
- High DeltaCp (10.7 +/- 0.7 kcal mol(-1) K(-1)) indicates a tightly packed tetramer; denaturant unfolding is irreversible.
- Free thiol reactivity is salt-dependent, suggesting proximity of cysteines.
Conclusions:
- SecB is a stable, well-folded tetramer.
- Substrate binding likely occurs at a surface site.
- Thermodynamic and structural data provide insights into SecB's chaperone function.