Single molecule study on protein dynamics by fluorescence confocal microscopy

Single molecule fluorescence spectroscopy provides a powerful tool to investigate molecular structural change and dynamics of heterogeneous biological system. This thesis work presents several projects involving experimental exploration and theoretical treatment based on statistical analysis. In single molecule optical research, two major challenges have to be overcome to obtain more reliable and meaningful information. One is the immobilization of singe molecules and the other is the analysis of limited data from stochastic single molecule signals. In this thesis, sol-gel encapsulation and trehalose immobilization have been explored and discussed, and they showed great promise to serve as good immobilization matrix. In another project, rotational dynamics of single Zinc Cytochrome C and single Porphyrin Cytochrome C were studied by two-channel polarization confocal microscopy. The results showed that proteins like Cytochrome C sensed very heterogeneous local environment within trehalose matrix and some of them actually were undergoing slow rotational motions. In the work of fluorescence lifetime distribution undergoing Förster energy transfer, single molecule technology was demonstrated to be very useful to solve inverse Laplace transform problem in a direct way. In the work of single CaM:peptide complexes, the distribution of fluorescence lifetime of probing dye, Nile Red, showed significant dependence of the calcium ion concentration, and the PAT analysis showed structural fluctuation of milli-second time scale at the mid-point of calcium ion titration curve. These results help to understand structural dynamics of CaM:peptide complex