Untersuchung, Entwicklung und Anwendung reversibel schaltbarer fluoreszierender Proteine

Abstract

Over the course of the last decade fluorescent proteins became an essential part of biological research. Since their first use as a marker for proteins in living cells, numerous fluorescent proteins in almost all colours of the visible spectrum with miscellaneous properties have been developed. A new subclass of these fluorescent proteins may be switched on and off between a fluorescent and a non-fluorescent state. These reversibly switchable fluorescent proteins (RSFPs) have great potential in the development and use of microscopic techniques because of their unique switching property. In this thesis the molecular mechanism of the reversible switching in RSFPs was identified. Furthermore, novel RSFPs with improved properties were generated and applied in different microscopy schemes. The molecular basis of switching was solved by x-ray analysis. Protein crystals were analysed after switching them completely into either the fluorescent or the non-fluorescent state to identify structural differences on the molecular level. The determination of these states for asFP595 and Dronpa, the only two known RSFPs at that moment, revealed a cis-trans-isomerisation of the chromophore as the key event of reversible switching. The isomerisation from the cis- into the trans-conformation changes the environment of the chromophore, resulting in different absorption and fluorescence properties. These differences arise from modifications in protonation and stabilisation of the two states. On the basis of these results, it was possible to propose a reduced model for the reversible switching, which holds for all currently known RSFPs. In order to customise the proteins, a screening setup was built, allowing for the directed evolution of RSFPs. By using random and directed mutagenesis the proteins rsFastLime, bsDronpa, Padron and Padron* were developed. These proteins exhibit improved switching properties and were especially designed for the use in high resolution microscopy schemes. For rsFastLime and bsDronpa, microscopy with a resolution beyond the diffraction limit was readily demonstrated. In addition to the use of RSFPs in high resolution microscopy, a new method allowing for multicolour imaging without aberrations, the monochromatic multilabel microscopy, was established. By solving the basic molecular mechanism of reversible switching and the subsequent development of new improved RSFPs, this thesis should broaden the possibilities for research in living cells.