Color Hues in Red Fluorescent Proteins Are Due to Internal Quadratic Stark Effect

Intrinsically fluorescent proteins (FPs) exhibit broad variations of absorption and emission colors and are available for different imaging applications. The physical cause of the absorption wavelength change from 540 to 590 nm in the Fruits series of red FPs has been puzzling because the mutations that cause the shifts do not disturb the π-conjugation pathway of the chromophore. Here, we use two-photon absorption measurements to show that the different colors can be explained by quadratic Stark effect due to variations of the strong electric field within the β barrel. This model brings simplicity to a bewildering diversity of fluorescent protein properties, and it suggests a new way to sense electrical fields in biological systems.