Fig 6. Time evolution of non-hydrogen atoms RMSD of residues 1-14 with respect to the unfolded starting structure and classification into quenched and fluorescent states in the time frame between 27.5–28.0 μs.

Although the backbone was mostly locked in cluster #1 conformation during this time span, spontaneous unstacking of Atto655/Trp15 was observed. Two exemplary structures shortly before and after an unstacking event are shown below. The stacked (grey) configuration quenches the Atto655 fluorescence, while the unstacked (red) configuration allows fluorescence. Unstacking was observed to occur on the subnanosecond timescale and is not coupled to a noticable change of the overall RMSD in this cluster.