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High-pressure denaturation of apomyoglobin
S E Bondos1, S Sligar, J Jonas
1Department of Biochemistry, Beckman Institute, University of Illinois, Urbana, IL 61801, USA.
Biochimica Et Biophysica Acta
|September 27, 2000
Summary
High pressure reveals apomyoglobin (apoMb) unfolding to a stable intermediate, similar to acid-unfolded states. Mutations destabilize apoMb, creating this intermediate at lower pressures, suggesting conserved folding pathways.
Area of Science:
- Protein dynamics and stability
- Biophysical chemistry
- Structural biology
Background:
- Apomyoglobin (apoMb) is a model protein for studying folding mechanisms.
- Understanding protein intermediates is crucial for deciphering folding pathways.
- Pressure-induced denaturation offers a unique tool to probe protein stability.
Purpose of the Study:
- To investigate the pressure denaturation of wild type and mutant apomyoglobin (apoMb).
- To characterize the high-pressure intermediate state of apoMb.
- To compare pressure-induced intermediates with acid-induced intermediates and folding intermediates.
Main Methods:
- High-pressure, high-resolution nuclear magnetic resonance (NMR) spectroscopy.
- High-pressure fluorescence spectroscopy.
- Site-directed mutagenesis of apomyoglobin.
Main Results:
- Wild type apoMb denatures to a high-pressure intermediate (I(p)) between 80-200 MPa, retaining structural features like sequestered tryptophans and AGH core NOESY cross peaks.
- The I(p) state shares properties with the acid-induced intermediate (I(2)), suggesting conserved intermediate structures.
- Mutations disrupting AGH core packing destabilized apoMb, leading to intermediate formation at lower pressures compared to wild type.
Conclusions:
- The AGH core's stability contributes to intermediate formation.
- High-pressure intermediates share structural similarities with acid-induced and kinetic folding intermediates.
- Perturbation methods (pressure, mutation) yield similar intermediate states, supporting conserved folding pathways.
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