Flow and Stop Protocol for the Long-Time Observation of Fluorescence from Free Single Molecules: Application to the Time-Series Analysis of Protein Folding

Abstract

A new method was developed to detect fluorescence intensity signals quantitatively from single molecules diffusing freely in a capillary flow cell. A unique optical system based on a spherical mirror was designed so that it enables us to detect the fluorescence intensity quantitatively irrespective of the location of molecules in the capillary. In addition, the “flow and stop” control of the sample in the capillary can observe free single molecules for about several seconds, which is more than 1000 times longer than the observation time of a typical confocal method. Here, the method was applied to observe time series traces of the denatured state of iso1-cytochrome c labeled with a fluorescent dye. The analysis of the single-molecule traces based on the concept of local equilibrium state (LES) demonstrated that a large fraction of the traces was assigned to one large LES and two small non-LESs (that cannot satisfy the condition of local equilibrium). Furthermore, a significant fraction of the traces shows the transitions among the LES and the non-LESs. The fraction of the traces showing the transitions among the LES and the non-LESs in cytochrome c is larger than that of the traces of dye only. The difference can therefore be interpreted to arise from not the intensity fluctuation of the dye but the dynamics intrinsic to the protein. The long-time observation of a free single molecule is expected to reveal slow dynamic property of biological molecules, which could not be resolved by the other conventional methods.