Disentangling Chromophore States in a Reversibly Switchable Green Fluorescent Protein: Mechanistic Insights from NMR Spectroscopy.

The photophysical properties of fluorescent proteins, including phototransformable variants used in advanced microscopy applications, are influenced by the environmental conditions in which they are expressed and used. Rational design of improved fluorescent protein markers requires a better understanding of these environmental effects. We demonstrate here that solution NMR spectroscopy can detect subtle changes in the chemical structure, conformation, and dynamics of the photoactive chromophore moiety with atomic resolution, providing such mechanistic information. Studying rsFolder, a reversibly switchable green fluorescent protein, we have identified four distinct configurations of its