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Abstract

Reversibly photoswitchable uorescent proteins (RSFPs) are genetically engineered proteins that can be switched by light absorption between a uorescent ON state and a dark OFF state. Among other applications they allow to increase the resolution beyond the difraction limit in cell imaging by uorescence microscopy. RSFPs have extended the possibilities of uorescence microscopy and other biotechnological tools, but the development of their properties is still far from being rationally designed. Thus, there might be much room for improvement if we manage to understand the switching mechanisms. The switching mechanisms in several negative RSFPs is being elucidated but still under debate. In this thesis I extend the theoretical knowledge about the photoswitching of negative RSFPs by studying the excited-state potential energy surface of both the ON- and the OFF-state. I compare three RSFPs, namely IrisFP, Dronpa and a fast switching single mutant of Dronpa called Dronpa2 to search for the origin of their diferent switching quantum yields in the ON-state. For the OFF-state, the results of the combined quantum mechanics{molecular mechanics (QM/MM) calculations show that chromophore photoisomerization happens in its neutral form and is followed by ground state deprotonation. This is in agreement with a very recent ultrafast absorption spectroscopy study for IrisFP, and studies on Dronpa and other negative RSFPs. Although the experimental results in both proteins show the same steps, I found that they have diferent processes at the atomic level due to structural and electrostatical diferences, but leading to the same intermediates. This is in contrast to the ON-state where I get the same picture for the three proteins studied, identifying the conical intersection that quenches the uorescence and controls the photoswitching quantum yield. The major diference between the three proteins in terms of uorescence and photoswitching characteristics comes from the diferent sterical environment produced by the residue 159, which is a methionine in diferent isomers in IrisFP and Dronpa and a smaller threonine that allows a faster isomerization of the chromophore in Dronpa2.