Microsecond Barrier-Crossing Collapse in Cytochrome c Folding Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy.

Protein folding is initiated with a volume contraction called collapse. While the microsecond collapse of horse cyt c has been intensely studied, its molecular mechanism remains elusive. To characterize the structural change, we prepared three cyt c variants labeled with a fluorescent dye at distinct sites (D50, A83, and E104). We track their dynamics via FRET between the dye and the heme by two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), which has single-molecule sensitivity and sub-microsecond time resolution. The results reveal that the collapse exhibits two-state dynamics between structurally distributed ensembles, occurring on a time scale of tens of microseconds across all variants. This indicates a global contraction of the whole protein structure with a substantial free energy barrier. A slight delay observed for the D50 variant suggests the late folding of its associated loop. This study demonstrates the capability of 2D FLCS to characterize a small free-energy barrier in complex biophysical processes.