A Study On Folding And Function Of Metamorphic Protein Mad2 By Single-molecule Fluorescence Techniques

Mad2,a key protein at the spindle checkpoint?SAC?and,adopts two distinct native folds at equilibrium under physiological conditions,an open inactive form?O-Mad2?and a closed active form?C-Mad2?.Mad2 has been found to play an important role in tumorigenesis and progression,it is therefore an important targets for cancer treatment.The interaction between Mad2 and Cdc20 is important to SAC,where the unusual two-state behavior of Mad2 plays a crucial role in checkpoint signaling.However,the mechanism of the transition between two different conformations of Mad2 and the interaction between Mad2and Cdc20 remains unclear.Therefore,in order to further explore the mechanism of Mad2conformational transition and the interaction between Mad2 and Cdc20,we designed seven double-site mutants of Mad2 using wild-type Mad2 as template.Then we expressed,purified and labeled the mutant protein.The single-molecule fluorescence resonance energy transfer?sm-FRET?was then used to study the folding/unfolding kinetics of two different conformers of Mad2.In addition,the conformational changes of Mad2 itself during the interaction between Mad2 and Cdc20 were explored by sm-FRET,which provided the basis for further revealing the interaction mechanism between Mad2 and Cdc20.The main contents are as follows:?1?Seven mutants of Mad2 were successfully constructed in order to label the mutants by Alexa Fluor488?AF488?and Alexa Fluor 647?AF647?.The Mad2 mutant protein was prepared by using E.coli expression system.Two different conformers of Mad2,O-Mad2 and C-Mad2,were purified by Ni²⁺affinity chromatography,gel filtration desalting and anion exchange chromatography.After labeled by AF488 and AF647,the mutants were furtherly characteriaed by mass apectrum,fluorescence spectrum and UV-Vis absorption spectra,which indicte that the mutant was successfully labeled by AF488 and AF647.In this work,we provided experimental foundation for the further study on interconversion between two conformers of Mad2 by single molecule fluorescence.?2?Using sm-FRET study Mad2 transition between two different conformation and folding/unfolding kinetic process.The experimental results show that under physiological conditions,Mad2 has two different natural conformations and can transform each other.The denaturation experiments of guanidine hydrochloride with different concentrations of O-Mad2and C-Mad2 showed that the conformation of Mad2 was extended with the increase of the concentration of guanidine hydrochloride.When the guanidine hydrochloride concentration was 3.5,the Mad2-C106A mutant appeared in the intermediate state,the two Mad2conformations are transformed through intermediates.?3?The dynamic process of the conformational changes in Mad2 was studied by total internal reflection fluorescence microscopy?TIRF?.The Mad2 protein was immobilized on a glass slide,and the conformational change was detected by TIRF.The system of studying the conformational changes of Mad2 protein by TIRF was preliminarily established,which lays the foundation for further study on its conformational changes.?4?Using sm-FRET to study the interaction between Mad2 and Cdc20.It was found that,as Cdc20 concentration increases,Mad2 will form dimers and creates intermolecular FRET.The conformation of Mad2 monomers that make up the dimer does not change significantly.The mechanism of the interaction between Mad2 and Cdc20 was preliminarily discussed.