Question

6. What is the general approach to changing fluorescence spectrum of GFPs? explain briefly

Answer 1

GFPs is mainly used to monitor gene expressions. Utilizing the small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins.

Some proteins shows initial increase in fluorescence intensity upon application of pressure above ambient conditions.

Small blue shifts in the fluorescence spectra with increasing pressure were seen in all proteins under study, hinting at increased rigidity of the chromophore environment.  Thus changing fluorescence spectrum also changes the pressure, some exhibit strong and abrupt changes in their fluorescence spectra at certain pressures due to structural modifications of the hydrogen bonding environment of the chromophore. now for such proteins to indentify the clear 3 dimentional structures absorption and emission.

Fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. ​ spectral properties changes due to irradiation with light of a specific wavelength and intensity

Some of the yellow-shifted GFP mutants that exhibit a dramatically decreased fluorescence quantum yield with fluorescence losses more than 90% and extremely short excited state lifetimes have been interpreted in terms of internal conversion induced by torsional motion within the chromophore​ which is all induced by the fluorescence spectrum.

Therefore it is neccessary to set the proper fluorescence spectrum for GFPs to understand absorption emmision and 3D structure of particular protein thereby understanding the fluorescent properties