Simultaneous Determination of Two Subdomain Folding Rates Using the "Transfer-Quench" Method

Area of Science:

• Biochemistry
• Protein Dynamics
• Spectroscopy

Background:

• Protein folding mechanisms are complex due to context-dependent intramolecular contact formation rates.
• Site-specific labeling and ultrafast spectroscopy monitor folding but can perturb nearby structures.
• Studying multiple neighboring subdomains simultaneously in one mutant is desirable for resolving folding interdependence.

Purpose of the Study:

• To develop a novel method,
• Transfer-Quench
• , for measuring the formation rates of two structural elements using a single triple-labeled mutant.
• To apply this method to investigate the folding mechanism of the B domain of staphylococcal protein A.

Main Methods:

• Utilized Förster resonance energy transfer (FRET) combined with fluorescence quenching.
• Employed a single triple-labeled mutant with donor and acceptor probes at loop ends and a quencher at an α-helical element.
• Monitored folding via acceptor emission changes correlated with loop closure and helix formation.

Main Results:

• The Transfer-Quench method successfully measured the rates of two distinct structural element formations.
• In the B domain of staphylococcal protein A, loop closure (segment 14-33) and helix HII nucleation (segment 33-29) were found to occur at the same rate.
• Natural amino acids (Tyr, Trp, Cys) were used as probes, minimizing structural perturbations.

Conclusions:

• The Transfer-Quench method provides a robust approach to study the kinetics of multiple folding events simultaneously.
• The findings support the nucleation-condensation model for protein folding, as evidenced by the synchronous loop closure and helix nucleation.