The Setup Of Double-channel Single Molecule Fluorescence Microscope And The Dynamics Study Of Protein Interaction

Progressive exploration and scientific study of the nature of life has put forward new requirements on the traditional analysis, many biological reactions are too complex to fully comprehend through the use of conventional ensemble techniques. Single-molecule detection(SMD) can investigate the dynamical heterogeneities of life system via analyzing physical parameters as the function of time to get the distribution and characteristics of molecular dynamics. At present, SMD offers an effective means in scientific research fields such as biophysical,molecular biology, physics, chemistry, medicine and nanoscale materials. Specifically, single molecule fluorescence spectroscopy has been widely used in the study of the dynamic process of nucleic acids and proteins, tracking single particles over microns of distances, and deciphering the rotational motion of multi-subunit systems. One of the most powerful methods to study the conformational changes of biomolecular dynamics and the kinetic process of interactions is fluorescence resonance energy transfer(FRET).The protein-protein interaction plays an important role and is of significance in understanding the mechanism of function modulation in cell and the potential application of disease treatment. However the mutual recognition and the transition between conformational states of protein usually take place in microsecond, millisecond or even second time region, and demonstrate temporal and spatial heterogeneity. In this thesis, we select plant Glutathione Peroxidase(GPX) and Thioredoxin(TRX) as the interaction protein to study the connection between protein conformational dynamics and protein interaction at single molecule level, by using the home-built double channel single molecular total internal reflection fluorescence microscopic(TIRFM) as the main research method. The details are as follows:1. The setup of double-channel single molecule fluorescence microscope: According to the design and arrangement of our project, we have successfully setup a double channel total internal reflection fluorescence microscopy system, which can realize single molecule FRET investigation, single molecule tracking and dynamical analysis. The design idea and building procedure of the experimental system have been introduced, including the selection and debugging of each instrument accessories. The whole system has been optimization through the test analysis and inspection of the double fluorescence labeling of DNA, which have laid the foundation for the study of dynamic process of protein interaction at single molecule level.2. The study of interaction between glutathione peroxidase 3(At GPX3) and thioredoxin-h9(At TRX9) from Arabidopsis thaliana: We combined TIRFM with single molecule FRET technology to study the protein interaction between At GPX3 and At TRX9 from Arabidopsis thaliana. In ensemble study, by using fluorescence labeling technique and fluorescence spectroscopy, it has been observed an obvious FRET phenomenon between the two proteins, confirming the existence of interaction between At GPX3 and At TRX9. At the single molecule level, we employed the home-built TIRFM system to study the real-time interaction process by monitoring the fluorescence intensity changes of donor labeled At GPX3 and acceptor labeled At TRX9, and analyzing the statistical distribution of sm FRET efficiency. After the electron exchange between the two interaction proteins, the FRET efficiency presents three kinds of distribution, including a distinct intermediate state. Interestingly, the intermediate state demonstrates two kinds of FRET efficiency, 0.8 and 0.4, corresponding to two different spatial distance in protein separation process. With the analysis of the amino acid sequence and crystal structure of two interaction proteins, it could be concluded that there are two kinds protein interaction pathways for the involved electron transfer and the formation of disulfide bonds between At GPX3 and At TRX9.In summary, we have successfully setup the double channel single molecule TIRFM system and carried out the real time observation of the interaction dynamics between At GPX3 and At TRX9 by single molecule fluorescence technology. The performance of this study will be of significance in building up the scientific correlation between protein conformational dynamics and GPX-TRX interaction, and in elucidating the mechanism of signal transduction of plant cell in stress environment.