A Rapid Flow Mixer with 11-μs Mixing Time Microfabricated by a Pulsed-Laser Ablation Technique:  Observation of a Barrier-Limited Collapse in Cytochrome c Folding

Proteins with more than ∼100 residues usually demonstrate a rapid and significant collapse as the earliest event in their folding dynamics; however, the limit in the time resolution of available experimental methods precluded us from conducting a detailed analysis of the event. To investigate the collapse in a real time, a new rapid flow mixer was developed by using a femtosecond laser ablation technique, which achieved a mixing time of 11 μs. By using the developed mixer, the collapse dynamics of cytochrome c, initiated by pH jumps after the solution mixing, was investigated. The time domain within 100 μs, which was previously unobservable, was clearly resolved and demonstrated that the process can be approximated by a single-exponential process. The results support the presence of a single kinetic barrier in the initial collapse of cytochrome c.