TIME-RESOLVED STRUCTURAL STUDIES OF MTFP07 FLUORESCENCE PHOTOSWITCHING

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose continued structural studies on the basis of fluorescence photoswitching in mTFP0.7, a monomeric cyan fluorescent protein distantly related to the green fluorescent protein (GFP). mTFP0.7 is initially brightly fluorescent with a peak emission wavelength of 488 nm, however, rapid photoswitching to a nonfluorescent form occurs when the protein is illuminated with blue light (~ 450 nm)[1]. Spontaneous fluorescence recovery to nearly 100% is observed on a time scale of about 8 minutes at room temperature. Photoswitched mTFP0.7 can be very rapidly reactivated by exposure to 360-400 nm light. Structures of mTFP0.7 have been solved in the fluorescent and cryo-trapped nonfluorescent states[2]. These suggest a mechanism for the change in emission characteristics. In the fluorescent state, the chromophore is cis-coplanar (as found in the vast majority of brightly fluorescent proteins), however, in the dark nonfluorescent state, the chromophore is trans, highly twisted, protonated and partially disordered. We concluded that the latter three characteristics could each be argued to lower emission efficiency and taken together provide a compelling explanation for the complete loss of fluorescence. These characteristics provide a useful model for photoswitching, applicable to all photoswitchable fluorescent proteins described to date. Furthermore, a dramatic rearrangement of internal side chains suggests a ?lock and key? mechanism to explain the long life of the nonfluorescent state. The proposed time-resolved crystallographic studies on mTFP0.7 seek to reveal the basis for the photoswitching phenomenon.