The Screening And Application Of Photoconvertible Fluorescent Protein Probes For Super-resolution Imaging

Super-resolution fluorescent microscopy has become an essential tool to studybiological molecules, pathways and events in living cells, tissues and animals.Currently, some super-resolution imaging technologies have overcome the diffractionlimit. However, we are far away from molecular resolution (1-5nm), in whichindividual molecules in a macromolecular assembly can be resolved. There are twomain classes of FPs used in super-resolution imaging: those that convert from a darkstate to a bright fluorescent state (PA-FPs), and those that change fluorescentwavelength on irradiation (photoshiftable FPs(PS-FPs)). All known PS-FPsirreversibly shift their wavelength but PA-FPs can photoactivate either reversibly orirreversibly. If we improve the photochemical and photophysical characteristics of FPs,may be we become able to observe the biological phenomenon at molecular level.In the second chapter, we reported a fast and easy method for choosingappropriate RFP for labeling secretory membrane proteins. Kd is an importantparameter to assess the oligomerization in vitro. But the basic Kd value of currentlyused RFPs is not complete, we first offered the Kd value of commonly used RFPs.Many red fluorescent proteins prone to form an artificial punctate when connect tosecretory pathway proteins. The reason for this has not been elucidated. We confirmedthe exact localization of forming artificial punctate when RFPs attached to anenvironment sensitive membrane protein Orai1, and speculated that the pKa of FPsmay play a key role during the formation of bright artificial punctate. All in all,mOrange2is praised highly to label secretory pathway membrane protein.Third chapter is based on wide-field illuminatinon. The screening photostablefluorescent proteins are very important during longer wide-field imaging. All organicfuorophores undergo irreversible photobleaching during prolonged illumination.Although fuorescent proteins typically bleach at substantially slower rate than manysmall-molecular dyes, in many cases the lack of suffcient photostability remains animportant limiting factor for experiments requiring large number of images of singlecell. Based on the crystal structures of mEos2, we constructed several mutant libraries,in which the mutant sites were located around the chromophore. Luckily, we got amutant that had T58S A60Q of mEos3.2. The photostability of mEos3.2-SQ was abouttwo times as mEos3.2on the conventional fluorescence microscopy and laser scanning confocal microscopy.In the fourth chapter, the photoconvertible fluorescent protein that has lessspectrum-cross, which is obtained by combining fluorescent screening and software,would be very useful in dual-color PALM imaging. Crystal structures of mEos2allowed us to verify fulfillment of the interactions hypothesized to cause changes inthe spectrum, supporting their contribution to the observed spectrum changing. Werationally designed several mutant libraries in which we screened for increasing theemission of580nm or decreasing the emission of630nm mutant. There are two pe aksof mEos3.2, in which the main peak is580nm and the minor peak is630nm. Currently,we got F61C P141C T143R of mEos3.2that the emission of630nm were about2/3ofmEos3.2.