Red Fluorescent Protein-based Mechanical Sensor And Single Molecule Research On Enzymatic Reaction By SmFRET

It is not easy for people to do the research of activities in cells or proteins in vivo,the discovery of fluorescent proteins and fluorescent molecules helped us to solve the problem.Small changes in living organisms do not need intrusion detections any more,but can be converted to fluorescent signals to be observed more conveniently outside.Since the discovery of fluorescent proteins,it has been found that fluorescent proteins can be used as ion sensors to observe changes of ion concentration in the organism.However,we suggested that fluorescent proteins can also be used as mechanical sensors by controlling the P-barrel structure of the protein to observe the mechanical changes in vivo.For the fluorescent molecules,more and more people started to do single molecule researches using fluorescence resonance energy transfer.In this paper,we selected Holliday Junction structure to do the research.We embedded a cleavage site in Holliday Junction,if we attach donor and acceptor fluorophores to the ends of helices,we can study the enzymatic reactions through fluorescence resonance energy transfer.This system can also be used to do a lot of single molecule researches.In chapter 1,we mainly introduce the background of the entire article.First,we will basically introduce the knowledge of protein,such as the basic constituent,the spatial structure and the function of protein.Then we will briefly introduce the discovery of GFP,the spatial structure and the luminescence mechanism of fluorescent groups.Since the basic DNA modification technology used in this paper was PCR,we will introduce the principle,the reaction system and the process of normal,mutation and megaprimer PCR.Finally,we will introduce the principle of FRET,TIR-FM required for FRET observation and substrate modification process used in the experiment.In chapter 2,we will introduce the RFP-based mechanical sensors in detail.Red fluorescent proteins are preferable to other fluorescent proteins because of the lower phototoxicity,greater tissue penetration and the in vivo imaging advantages.We chose RFP to construct mechanical sensors,so that they can be used biologically in the future.We constructed mechanical sensors by linking ends to the N-terminal and C-terminal of RFP which had binding force to the P-barrel structure.In this way,we can change the fluorescence intensity of RFP.If we correspond the fluorescence intensity to the force applied to RFP,a mechanical sensor will be formed.In addition,we found that if the ?-barrel structure has great changes such as severe squeeze,the emission peak of the emission spectrum of the fluorescent protein will have blue shift,even the color of the fluorescent protein can be changed.Such changes can also be used to observe the mechanical changes in vivo.In chapter 3,we will introduce our system of observing enzymatic reactions.We chose restriction enzyme BamHI and embed its sequence into Holliday Junction.The research is based on the method of FRET to amplify the experimental results.Holliday Junction has two structures changes under certain conditions,which reacts on the efficiency of FRET.If the length of the branch of Holliday Junction changes,the fluctuation rate of the FRET efficiency between the two conformers will also change,which allow us to observe the kinetic characteristics of the enzymatic reactions well.Finally,we will summarize all the work above and propose future work which can be studied further.