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SecA folds via a dimeric intermediate
S M Doyle1, E H Braswell, C M Teschke
1Department of Molecular and Cell Biology and National Center for Analytical Ultracentrifugation, University of Connecticut, Storrs, Connecticut 06269-3125, USA.
Biochemistry
|September 20, 2000
Summary
SecA protein folding was studied using urea and fluorescence. Researchers found SecA unfolds via a dimeric intermediate before dissociating, revealing its stability and folding pathway.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Folding Dynamics
Background:
- Many cellular proteins are large and multimeric, yet their folding mechanisms remain understudied.
- SecA, a homodimeric protein crucial for bacterial protein translocation, is a relevant model due to its size and potential partial unfolding during membrane insertion.
Purpose of the Study:
- To investigate the folding pathway and stability of the bacterial protein SecA.
- To understand how SecA's structure relates to its function in preprotein translocation.
Main Methods:
- Kinetic and equilibrium folding monitored using circular dichroism and tryptophan fluorescence.
- Equilibrium folding also assessed with a fluorescent ATP analogue.
- Protein stability confirmed via analytical ultracentrifugation (equilibrium and velocity sedimentation).
Main Results:
- A three-state folding model was proposed: N(2) -> I(2) -> 2U, involving a dimeric intermediate.
- SecA dimer exhibits significant stability (ΔG = -22.5 kcal/mol).
- The rate-limiting step is the conversion of the dimeric intermediate to the native dimer; unfolding is slow.
Conclusions:
- SecA functions as a stable dimer, making monomer dissociation during translocation improbable.
- Understanding SecA's folding pathway provides insights into its essential role in the Sec-dependent translocase system.