Submillisecond protein folding kinetics studied by ultrarapid mixing

Area of Science:

• Biochemistry
• Physical Chemistry
• Molecular Biology

Background:

• Protein folding is crucial for biological function.
• Understanding protein folding dynamics, especially at early stages, remains challenging.
• Previous methods limited the study of rapid folding events.

Purpose of the Study:

• To develop and apply an ultrarapid-mixing continuous-flow method for studying submillisecond protein folding.
• To investigate the folding kinetics of cytochrome c in the microsecond to millisecond time range.
• To elucidate the initial events of protein folding, including collapse and barrier crossing.

Main Methods:

• Utilized an ultrarapid-mixing continuous-flow technique with turbulent flow for rapid denaturant dilution (tens of microseconds).
• Studied cytochrome c folding kinetics using the imidazole complex to prevent heme-ligand exchange complications.
• Monitored protein folding via fluorescence quenching (tryptophan to heme energy transfer) to track structural changes.

Main Results:

• Observed biphasic fluorescence decrease kinetics, indicating distinct folding phases.
• Identified an initial, rapid process (tau < 50 microseconds) likely representing protein collapse.
• Characterized a slower, exponential phase (tau = 600 microseconds) corresponding to free energy barrier crossing.

Conclusions:

• The developed method allows probing protein folding in previously inaccessible submillisecond timescales.
• Cytochrome c folding involves a rapid, barrier-free collapse followed by slower conformational changes.
• The findings provide new perspectives on the fundamental mechanisms of protein collapse and folding dynamics.