The reversible photoswitching between the ‘on’ and ‘off’ states of the fluorescent proteinDronpa involves photoisomerization as well as protein side-chain rearrangements, but theprocess of interconversion remains poorly characterized. Here we use time-resolved infraredmeasurements to monitor the sequence of these structural changes, but also of protontransfer events, which are crucial to the development of spectroscopic contrast. Light-induceddeprotonation of the chromophore phenolic oxygen in the off state is a thermal ground-stateprocess, which follows ultrafast (9 ps) trans–cis photoisomerization, and so does not involveexcited-state proton transfer. Steady-state infrared difference measurements exclude protonationof the imidazolinone nitrogen in both the on and off states. Pump–probe infraredmeasurements of the on state reveal a weakening of the hydrogen bonding between Arg66and the chromophore CO, which could be central to initiating structural rearrangement ofArg66 and His193 coinciding with the low quantum yield cis–trans photoisomerization
展开▼
