Single Molecule Fluorescence can Determine the Kinetics of the Transition

The solvent exposed (“out”) conformation is more fluorescent than the (“in”) sequestered conformation. The fluctuations in fluorescence are due to the switching from the out to the in conformation and back again.

A variety of controls is used to ensure the on-off signal is due to conformational changes and not due to photochemical effects or other reasons. The time spent in each state can then be used to calculate a rate by histogramming the data from a single molecule or several molecules.