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Trehalose favors a cutinase compact intermediate off-folding pathway
Eduardo P Melo1, LuYang Chen, Joaquim M S Cabral
1Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal. emelo@ualg.pt
Biochemistry
|June 18, 2003
Summary
Trehalose stabilizes compact states of cutinase during folding and unfolding. This compatible solute stabilizes an intermediate that appears off the folding pathway, affecting enzyme kinetics.
Area of Science:
- Biochemistry
- Protein Folding Dynamics
- Enzyme Kinetics
Background:
- Cutinase is an enzyme with lipolytic activity.
- Trehalose is a compatible solute known to stabilize proteins.
- Understanding protein folding pathways is crucial for enzyme function.
Purpose of the Study:
- To investigate the effect of trehalose on cutinase folding and unfolding.
- To characterize the intermediates involved in the cutinase folding pathway.
- To elucidate the role of trehalose in stabilizing compact conformational states.
Main Methods:
- Equilibrium unfolding experiments.
- Kinetic studies of protein folding and unfolding.
- Analysis of chevron plots to determine folding intermediates and transition states.
Main Results:
- Trehalose increases the free energy difference between folded and unfolded cutinase.
- Unfolding kinetics reveal a partially folded intermediate (approx. 60% solvent exposure) stabilized by trehalose.
- Folding kinetics identify a compact intermediate, stabilized by trehalose, which appears to be off the main folding pathway.
Conclusions:
- Trehalose stabilizes compact conformational states of cutinase, including intermediates.
- The stabilizing effect of trehalose on compact states aligns with models of compatible solute function.
- Compact intermediates that decelerate folding are also stabilized by trehalose, impacting enzyme folding kinetics.