Time-resolved Fluorescence Dynamics Of Proteins And Its Application On NADH/NAD~+ Ratiometric Quantification

Study on protein structure and function has been academic hotspot of modern biology for decades.Fluorescence techniques are widely used in this field because of its sensitivity and ability to detect biochemical interactions at molecular scale.Time-resolved fluorescence technique plays an important role in fluorescence detection.Compared with steady-state fluorescence,time-resolved method shows higher sensitivity and resolution,which have gained considerable attention and development in recent years.This dissertation is mainly focused on biological topics researched by time-resolved fluorescence method.Fluorescence dynamics of LicT proteins and SNase variants were studied and the protein structure changes were discussed.Using the same experimental methods and theoretical principles,a novel sensitive method for NADH/NAD+ quantification by genetically encoded fluorescent biosensors was proposed based on the time-resolved fluorescence dynamics.At the same time,pH-effect free measurement for cpYFP based NADH sensor was also developed by time-resolved method.The first part of this dissertation explored time-resolved fluorescence properties of two protein systems:LicT proteins and SNase variants.Firstly,the fluorescence dynamics of tryptophan residues in LicT proteins was investigated by time-resolved fluorescence method combined with UV absorption and steady-state fluorescence spectroscopy.The local microenvironment and structural changes of LicT protein before and after activation were studied.The activated LicT protein AC 141 prevents the antitermination of gene transcription involved in carbohydrate utilization to accelerate the body's metabolism.The structural properties and microenvironment of activated protein AC 141 and wild-type protein Q 22 were determined by fluorescence emissions and lifetimes of tryptophan residues.The interaction between tryptophan residues and solvent is elucidated by decay associated spectroscopy(DAS)and time-resolved emission spectra(TRES),indicating that upon activation,the structure of AC 141 is more compact than that of wild-type Q 22.In addition,TRES also showed that tryptophan residues in the protein had a continuous spectral relaxation process.Anisotropy results illustrated the conformational motions of residues and whole proteins,suggesting that tryptophan residues had independent local motions in the protein system,and that the motions were more intense in the activated protein.Secondly,the fluorescence dynamics of tryptophan residues of two staphylococcal nuclease(SNase)variants ?+PHS and ?+PHS+I92A were studied by time-resolved fluorescence method combined with UV absorption spectroscopy and steady-state fluorescence spectroscopy.The steady-state fluorescence spectra of the two variants showed the protective effect of SNase helix structure on tryptophan residue.The different changing trends of decay-associated spectra(DAS)upon temperature indicated that the two variants were different in folding and thermal stability.The continuous relaxation process of SNase variants was confirmed by the continuous spectral relaxation process of 0.5 ns of tryptophan residues in time-resolved emission spectra(TRES).The analyses of upconversion data showed the effect of relaxation on tryptophan residues.Anisotropy parameters showed the conformational motion of the Trp residues and the entire proteins.The results indicated that the tryptophan residues had independent local motion in the protein system and its intensity was related to the overall effect of thermal stability and thermal motion of the SNase variants.Thus,tryptophan could be used as an endogenous probe to confirm and study the folding structure and thermal stability of SNase variants.In addition,these conclusions help researchers use time-resolved fluorescence methods to further investigate structural dynamics of other SNase variants.On the basis of time-resoved fluorescence principles and techniques above,the second part of this dissertation put forward a highly sensitive NADH/NAD+ratiometric quantification method which may provide a new detection of redox state of living cells.Time-resolved fluorescence of genetically encoded fluorescent NADH/NAD+ biosensors Peredox,SoNar,and Frex were studied.When the NADH/NAD+ redox state changes,the lifetime components(?i)stay nearly constant,whereas their respective fractional amplitudes(?i)change.Most importantly,due to the conformation changes between two different states of the sensors,the fractional intensities(?i?i)have opposite changing trends in terms of NADH binding.Their ratios could be exploited to quantify NADH/NAD+levels with a larger dynamic range and higher resolution than that of-the commonly used fluorescence intensity and average lifetime methods.Moreover,only one excitation and one emission wavelength are required for this ratiometric measurement.This research elucidates a novel method to simplify the design and achieve highly sensitive analyte quantification of genetically encoded fluorescent biosensors.Wide applications could be developed for imaging live cell metabolism based on this new method.What's more,pH effect free measurement for cpYFP based NADH sensor was also developed based on the similar time-resolved method.It helps to broaden the application of cpYFP sensors on live cell imaging.In this dissertation,time-resolved fluorescence techniques were used to study the dynamic process of protein systems and to develop a novel NADH/NAD + ratiometric quantificaiton method based on fractional intensities of genetically encoded biosensors.At first,structural changes of LicT proteins upon activation and folding and thermal stability of SNase variants were discussed,which provided a new idea for study of protein structure and function.On the basis of previous research,the fluorescence dynamics of NADH/NAD+ fluorescent biosensors was studied,and a highly sensitive time-resolved NADH/NAD+ quantification method was proposed.Besides,pH effect free measurement for cpYFP based NADH sensor was also developed based on the similar time-resolved method.All these novel methods provide new methods for imaging of redox state in live cells.