ULTRA-FAST EXCITED-STATE DYNAMICS IN GREEN FLUORESCENT PROTEIN - MULTIPLE STATES AND PROTON-TRANSFER

The green fluorescent protein (GPP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromo phore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit. This permits fusion of the DNA s equence of GFP with that of any protein whose expression or transport can then be readily monitored by sensitive fluorescence methods withou t the need to add exogenous fluorescent dyes. The excited state dynami cs of GFP were studied following photoexcitation of each of its two st rong absorption bands in the visible using fluorescence upconversion s pectroscopy (about 100 fs time resolution). It is shown that excitatio n of the higher energy feature leads very rapidly to a form of the low er energy species, and that the excited state interconversion rate can be markedly slowed by replacing exchangeable protons with deuterons, This observation and others lead to a model in which the two visible a bsorption bands correspond to GFP in two ground-state conformations. T hese conformations can be slowly interconverted in the ground state, b ut the process is much faster in the excited state. The observed isoto pe effect suggests that tile initial excited state process involves a proton transfer reaction that is followed by additional structural cha nges. These observations may help to rationalize and motivate mutation s that alter the absorption properties and improve the photo stability of GFP.