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To twist or not to twist: From chromophore structure to dynamics inside engineered photoconvertible and photoswitchable fluorescent proteins

Green‐to‐red photoconvertible fluorescent proteins (FPs) are vital biomimetic tools for powerful techniques such as super‐resolution imaging. A unique Kaede‐type FP named the least evolved ancestor (LEA) enables delineation of the evolutionary step to acquire photoconversion capability from the ancestral green fluorescent protein (GFP). A key residue, Ala69, was identified through several steady‐state and time‐resolved spectroscopic techniques that allows LEA to effectively photoswitch and enhance the green‐to‐red photoconversion. However, the inner workings of this functional protein have remained elusive due to practical challenges of capturing the photoexcited chromophore motions in real time. Here, we implemented femtosecond stimulated Raman spectroscopy and transient absorption on LEA‐A69T, aided by relevant crystal structures and control FPs, revealing that Thr69 promotes a stronger π–π stacking interaction between the chromophore phenolate (P‐)ring and His193 in FP mutants that cannot photoconvert or photoswitch. Characteristic time constants of ~60–67 ps are attributed to P‐ring twist as the onset for photoswitching in LEA (major) and LEA‐A69T (minor) with photoconversion capability, different from ~16/29 ps in correlation with the Gln62/His62 side‐chain twist in ALL‐GFP/ALL‐Q62H, indicative of the light‐induced conformational relaxation preferences in various local environments. A minor subpopulation of LEA‐A69T capable of positive photoswitching was revealed by time‐resolved electronic spectroscopies with targeted light irradiation wavelengths. The unveiled chromophore structure and dynamics inside engineered FPs in an aqueous buffer solution can be generalized to improve other green‐to‐red photoconvertible FPs from the bottom up for deeper biophysics with molecular biology insights and powerful bioimaging advances.