The photochemistry of fluorescent proteins: implications for their biological applications

Green fluorescent protein from Aequorea victoria, its relatives and derivatives are ubiquitous in their use as biological probes. In this tutorial review, we discuss the photochemistry of this fascinating class of proteins and illustrate some of their advantages and drawbacks in a range of applications. In particular, we focus on the ionisation states of the chromophore and how they are affected by internal and external proton transfer. Light-induced reversible and irreversible events are discussed in terms of the underlying chromophore structure. These phenomena have an influence on the interpretation of FRET (Forster resonance energy transfer), FRAP (fluorescence recovery after photobleaching), as well as single molecule studies.