Related Experiment Videos
The A-state of barnase
J M Sanz1, C M Johnson, A R Fersht
1MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, Medical Research Council Centre, U.K.
Biochemistry
|September 20, 1994
Summary
Acid denaturation of barnase reveals partly-folded intermediates. Mutants accumulating folding intermediates form soluble aggregates (A-state) at low pH, suggesting intermediates stabilize and self-associate.
Area of Science:
- Biochemistry
- Protein Folding
- Biophysical Chemistry
Background:
- Barnase denaturation at low pH deviates from two-state behavior.
- This deviation is pH-dependent and influenced by ionic strength.
- Specific barnase mutants exhibit complex thermal denaturation profiles.
Purpose of the Study:
- Investigate acid-induced denaturation of barnase and its mutants.
- Identify and characterize partly-folded protein intermediates.
- Explore the relationship between folding intermediates and aggregation.
Main Methods:
- Differential scanning calorimetry (DSC) of barnase and mutants.
- Far-UV circular dichroism (CD) spectroscopy.
- Kinetic and chemical cross-linking experiments.
Main Results:
- Barnase denaturation deviates from two-state behavior below pH 4.0, restored by KCl.
- Mutants accumulating folding intermediates show complex thermal denaturation.
- A soluble aggregated state (A-state) with molten globule-like properties forms, particularly in mutants.
- Dimerization of partly-folded molecules is likely rate-limiting for A-state formation.
Conclusions:
- The A-state is related to barnase's main folding intermediate.
- This intermediate is stabilized at low pH and prone to self-association.
- Partly-folded intermediates can lead to aggregation under specific conditions.