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Favourable native-like helical local interactions can accelerate protein folding
A R Viguera1, V Villegas, F X Avilés
1European Molecular Biology Laboratory, Heidelberg, Germany.
Folding & Design
|January 1, 1997
Summary
Stabilizing proteins through local interactions enhances thermoresistance and can increase folding speed, challenging theories that favor nonlocal interactions for optimal folding. This approach was demonstrated in ADA2h protein mutants.
Area of Science:
- Protein biophysics
- Molecular biology
- Biochemistry
Background:
- Protein stability is influenced by helix stability, which can be enhanced through local interactions to resist denaturation.
- Studies on Escherichia coli CheY and human procarboxypeptidase A2 activation domain (ADA2h) provide insights into protein folding mechanisms.
Purpose of the Study:
- To investigate the impact of stabilizing alpha-helices in ADA2h using local interactions on protein folding kinetics and stability.
- To determine if optimizing folding speed requires a balance favoring nonlocal interactions, as suggested by theoretical models.
Main Methods:
- Engineering of ADA2h mutants (M1, M2, DM) with stabilized alpha-helices through local interactions.
- Analysis of equilibrium and kinetic behavior using chemical denaturation (urea, GdnHCl) and refolding/unfolding assays.
Main Results:
- The double mutant (DM) exhibited significantly enhanced stability and thermoresistance (Tm > 363 K), being ~2.6 kcal mol-1 more stable than wild type (WT).
- Single mutants showed varied effects: M1 had decelerated unfolding, while M2 displayed both decelerated unfolding and accelerated refolding.
- The refolding slope (mkf) was reduced in M2 and DM mutants compared to WT and M1, indicating altered folding kinetics.
Conclusions:
- Thermoresistance can be achieved by increasing favorable native local interactions, even altering the local/nonlocal interaction balance.
- Mutational redesign introducing local interactions can accelerate protein folding, contradicting theories prioritizing nonlocal interactions for speed.
- These findings, demonstrated in ADA2h, suggest that not all proteins are optimized for rapid folding and that effects may differ in proteins with residual structure or complex denatured states.