Submillisecond folding of the peripheral subunit-binding domain.

Area of Science:

• Biochemistry
• Structural Biology
• Chemical Physics

Background:

• Protein folding rates are crucial for understanding protein function and stability.
• Traditional methods like stopped-flow are insufficient for measuring extremely fast folding kinetics.
• The peripheral subunit-binding domain is a small, three-helix protein relevant to biological processes.

Purpose of the Study:

• To accurately measure the folding and unfolding rates of the peripheral subunit-binding domain.
• To investigate the influence of temperature on these folding kinetics.
• To correlate folding rates with protein topology and compare with known folding speeds.

Main Methods:

• Dynamic Nuclear Magnetic Resonance (NMR) lineshape analysis was employed to measure folding and unfolding rates.
• Measurements were conducted across a range of temperatures, focusing on temperatures with sufficient spectral broadening.
• Specific resonances from Val16 and Val21 were monitored to ensure rate accuracy.

Main Results:

• The peripheral subunit-binding domain folds extremely rapidly, with rates too fast for stopped-flow techniques.
• At 41°C, the folding rate was measured at 16,050 s⁻¹, resulting in a folding halftime of 43 microseconds.
• The unfolding rate at the same temperature was 2800 s⁻¹, with identical rates confirmed using different NMR resonances.

Conclusions:

• The peripheral subunit-binding domain is the third fastest-folding protein documented and possesses the fastest directly measured folding rate.
• The rapid folding kinetics are consistent with its protein topology.
• These findings underscore the significant role of chain topology in determining protein folding rates.