Protein Hydrophobic Collapse and Early Folding Steps Observed in a Microfluidic Mixer

We demonstrate that the sub-millisecond protein folding process referred to as “collapse” actually consists of at least two separate processes. We observe the UV fluorescence spectrum from naturally occurring tryptophans in three well-studied proteins, cytochrome c, apomyoglobin, and lysozyme, as a function of time in a microfluidic mixer with a dead time of ∼20 μs. Single value decomposition of the time-dependent spectra reveal two separate processes: 1), a spectral shift which occurs within the mixing time; and 2), a fluorescence decay occurring between ∼100 and 300 μs. We attribute the first process to hydrophobic collapse and the second process to the formation of the first native tertiary contacts.