Three photoconvertible forms of green fluorescent protein identified by spectral hole-burning

Several studies have led to the conclusion that, in the green fluorescent protein (GFP) of the jellyfish Aequorea victoria, a photoconversion involving excited-state proton transfer occurs from an A- to a B-form, while an intermediate I-form was held responsible for the green fluorescence. Here we have identified the I-form of wild-type GFP in absorption, located the 0-0 transitions of all three forms A, B and I, and determined vibrational frequencies of the ground and excited states. The intrinsically narrow 0-0 transitions are revealed by the wavelengths at which holes can be burnt. The pathways of photointerconversion are unraveled by excitation, emission and hole-burning spectroscopy. We present an energy-level scheme that has significant implications for GFP-mutants, which likewise can occur in the three photo-interconvertible forms.