Ultrafast Dynamics And Mechanism Of Bilin-based Fluorescent Proteins

Fluorescent labeling has realized the spatiotemporal visualization of cells,tissues and living organisms,and greatly contributes to the development of life sciences such as modern biology and medicine.As the key in labeling,fluorescent proteins(FPs)are required to possess excellent spectral properties and wide applications.In recent years,bilin-based FPs incorporate with linear tetrapyrroles as chromophores have become new directions in the development of fluorescent labeling.On the one hand,they show short maturation time,oxygen-independence and no hydrogen peroxide production during expression and imaging,since their chromophore bilirubin(BR)and biliverdin(BV)are naturally present in mammalian cells as the degradations of heme.On the other hand,BV-based NIR FPs can be applied in deep tissue imaging within optical imaging window(650-900 nm)which shows low interference from absorption,autofluorescence and background scattering.However,the studies on bilin-based FPs begin at last few years,and the developments for these FPs are not as good as traditional GFP super families.In order to have a better understanding of the mechanism and to help with the development of these bilin-based FPs,steady-state spectroscopic measurements and ultrafast time-resolved spectroscopic mesurements were performed on these bilin-based FPs from three different origins.We studied their excited state dynamics and mechanism,and revealed their time-resolved spectral properties and the key factors in determining fluorescence quantum yield.Thus,this study provides important mechanism basis to further develop the bilin-based FPs with better spectral properties for labeling or functions for biosensing.The main researches of this thesis are as follows:1.Femtosecond transient absorption spectroscopy(TA)and time-dependent density functional theory calculation(TD-DFT)were used to study the molecular conformation of bilirubin in different solvents,and the relationship between fluorescence emission and the distribution of electron cloud in molecular orbital,and the relationship between the excited state lifetimes of bilirubin and solvent viscosities,and the excited state proton transfer process.As a result,the excited state dynamics of bilirubin in solutions were revealed and the mechanism of fluorescence emission and the dissipation pathway of excited state energy were obtained.2.In order to develope BR-based fluorescent probe,cyclodextrins,quinine,human serum albumin,and various metal ions were selected to interact with bilirubin to improve the solubility and fluorescence QY of bilirubin in water.Their interactions were investigated,and the potential applications were also proposed.3.Picosecond time-resolved fluorescence spectroscopy and femtosecond transient absorption spectroscopy were performed on green fluorescent protein UnaG,which comes from vertebrates,and several mutants at different sites to study their excited state dynamics.With these time-resolved experiments,isotope experiments,and the analysis of crystal structure of wt-UnaG and its mutants,we obtained the key factors in determining the fluorescence quantum yield,excited state lifetime,structural homogeneity in the ground state as well as emission dynamics of bilirubin in UnaG.4.Picosecond time-resolved fluorescence spectra and femtosecond transient absorption spectra were also used to study the excited state dynamics of the miRFP670 engineered from bacterial phytochrome(Bphs)and smURFP engineered from cyanobacterial phycobiliprotein,and the corresponding dynamics models were obtained.In addition,the radiative decay rates,non-radiative decay rates and exicted state decay rates were calculated for currently reported BV-based FPs,and a new direction to develop these FPs was proposed.