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Cooperative sub-millisecond folding kinetics of apomyoglobin pH 4 intermediate
Sebastien Weisbuch1, Francine Gérard, Marielle Pasdeloup
1Laboratoire de Biophysique Moléculaire et Cellulaire, Université Joseph Fourier, BMC/DRDC, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.
Biochemistry
|May 4, 2005
Summary
Protein folding is a cooperative process. All steps in apomyoglobin folding, including early intermediates, occur as two-state reactions, involving simultaneous secondary structure formation and hydrophobic collapse.
Area of Science:
- Protein folding dynamics
- Biophysical chemistry
- Molecular biology
Background:
- Small single-domain proteins typically fold via cooperative processes, often modeled as two-state transitions.
- The cooperativity of early folding steps and intermediates remains less understood.
Purpose of the Study:
- To investigate the cooperativity of each step in the apomyoglobin folding pathway.
- To determine if folding intermediates are formed through highly cooperative processes.
Main Methods:
- Kinetic measurements of apomyoglobin refolding and unfolding using far-UV circular dichroism (CD) and Trp fluorescence.
- Stopped-flow spectroscopy to analyze sub-millisecond folding kinetics.
Main Results:
- The earliest folding step (U <=> Ia) exhibits cooperative kinetics, with secondary structure formation and hydrophobic collapse occurring concurrently.
- Subsequent folding steps (Ia <=> Ib and Ib <=> N) also demonstrate cooperative, two-state reaction characteristics.
- Kinetics measured by CD and Trp fluorescence were consistent across different folding stages.
Conclusions:
- Each identified step in apomyoglobin folding, including early intermediates, proceeds as a highly cooperative, two-state reaction.
- These findings clarify the cooperative nature of protein folding pathways, even at early stages.