Fast folding of a four-helical bundle protein

Area of Science:

• Protein folding dynamics
• Biophysics of protein structure
• All-helical protein kinetics

Background:

• The FK506-FKBP12 binding domain (FRB) of kinase FRAP is an up-down four-helical bundle.
• Understanding protein folding pathways is crucial for molecular biology and drug development.

Purpose of the Study:

• Investigate the folding pathway of the FRB protein.
• Characterize the nature and role of any intermediate states during FRB folding.
• Compare FRB folding kinetics with other all-helical proteins.

Main Methods:

• Combined equilibrium and kinetic studies.
• Chemical denaturation analyzed using rate constants.
• Refolding experiments with hydrophobic dye 8-anilinonaphthalene-1 sulfonate (ANS).
• Stopped-flow fluorescence detection of ANS binding to intermediates.

Main Results:

• FRB native state is stable (7 kcal mol(-1) at pH 6.0, 10°C).
• A compact intermediate state, possibly misfolded, is populated during folding at low denaturant concentrations.
• ANS binding confirms the intermediate state, which undergoes fluorescence changes detected by stopped-flow.
• Folding and unfolding rates in water are ~150-200 s(-1) and 0.005-0.06 s(-1) respectively.
• The transition state for folding is compact, with ~80% of unfolded surface area buried.

Conclusions:

• FRB folding is slowed by a metastable intermediate, though it still folds rapidly (5 ms half-life at 10°C).
• The intermediate may require partial unfolding before reaching the transition state.
• FRB folding kinetics provide insights into all-helical protein folding mechanisms.