Fluorescence Kinetics Of Genetically Encoded Histidine Probe FHisJ And Its Applications

The monitoring of life activities has always been a hot issue in the field of life sciences,and the realization of this goal depends on the continuous development of exploration tools.Fluorescent protein biosensors have been developed as a powerful tool in the field of life sciences for decades.Compared with chemical fluorescent dyes,fluorescent protein biosensors are more biocompatible and less toxic.And it can express in the cell more accurately as it is easy to genetically encode.As a way to construct fluorescent protein probes,circularly permuted fluorescent protein(cp FP)biosensors are more susceptible to external analytes though they do not change the threedimensional structure of the original fluorescent protein,making cp FP biosensors own larger dynamic range and higher sensitivity.Now it has received more and more attention.FHis J,with specific response to histidine,was selected as a research object to study circularly permuted yellow fluorescent proteins(cp YFPs)in this paper.By using timeresolved fluorescence and steady-state fluorescence techniques,the mechanism of cp YFPs were investigated.The energy level structure of the cp YFPs and the change of the energy level structure after the addition of the analytes were proposed.Then another cp YFP biosensor,named So Nar,which responded to NADH/NAD+,was selected to verify the accuracy of our model.At the same time,we proposed new standards to measure the concentration and other information of analytes,which was applied in the detection of histidine by using FHis J,and obtained a large dynamic range.This method also can be applied successfully to living cells.In the end,we explored the reasons for the little change in the average fluorescence lifetime of cp FPs.Our work provides some suggestions for the design of better biosensors based on cp FPs in the future.