In-vitro-Untersuchung einzelner fluoreszierender Proteine mit spektral und zeitlich aufgelöster Fluoreszenzspektroskopie

This work presents spectroscopic analyses of the EGFP and EYFP variants of the Aequorea green fluorescent protein (GFP) and of the red fluorescing protein DsRed and four of its variants from the coral Discosoma. In addition to ensemble spectroscopy, the fluorescence of a large number of single fluorescing proteins and protein oligomers was temporally- and spectrally- resolved at the single molecule level in vitro at room temperature.
These analyses reveal that at the single molecule level these proteins exhibit a striking intrinsic versatility and dynamics of the emission. It was shown that the spread of the distribution of the emission maximum positions is characteristic for each variant and directly reflects the specific nature of the embedding of the chromophore into its neighbouring protein environment. It was further possible to identify different emitting forms of the proteins, some not observed until now, and to follow transitions between the forms by spectrally resolving the single molecule emission.
The typical conditions for single molecule detection employed in these experiments, especially the high excitation power, caused an extremely rapid transition of the matured chromophore of DsRed and all of the tested variants into a ‘super-red’ emitting form. Mixed fluorescence of the immature and the mature chromophore was detected for all DsRed variants, yielding evidence of the existence of mixed oligomers of proteins with mature and immature chromophores. Finally it was possible to show that the different chromophores within an oligomer do not form a efficient FRET system.