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The alpha-helix folds on the millisecond time scale
D T Clarke1, A J Doig, B J Stapley
1Daresbury Laboratory, Daresbury, Warrington, Cheshire WA4 4AD, United Kingdom.
Summary
Helix nucleation, previously thought to be extremely fast, actually occurs on the millisecond timescale. This study reveals that alpha-helix formation is significantly slower than anticipated, impacting protein folding dynamics.
Area of Science:
- Biochemistry
- Biophysics
- Physical Chemistry
Background:
- The nucleation of alpha-helices is a critical early step in protein folding.
- Previous estimates suggested helix nucleation occurs within nanoseconds (10(-7) s).
Purpose of the Study:
- To directly measure the timescale of alpha-helix nucleation.
- To investigate the kinetics of helix formation in peptides and homopolymers.
Main Methods:
- Utilized stopped-flow deep UV circular dichroism (CD) spectroscopy.
- Employed synchrotron radiation for enhanced signal-to-noise ratio CD measurements.
- Studied polyalanine-based peptides and lysine/glutamic acid homopolymers.
Main Results:
- Alpha-helix nucleation was found to occur on the millisecond timescale, significantly slower than previously believed.
- A 16-amino acid AK peptide exhibited first-order kinetics with a rate constant of 15 s(-1) at 0°C.
- Longer peptides showed multi-step folding kinetics with a transient overshoot in helix content.
Conclusions:
- Helix nucleation is a rate-limiting step occurring on the millisecond timescale.
- The observed folding overshoot suggests a mechanism involving initial formation of a single helix followed by fragmentation.
- Findings challenge existing models of rapid alpha-helix initiation and protein folding.