Development of the Line Confocal System for the Single Molecule Tracking of Fast Folding Dynamics of Proteins

Abstract

Single-molecule fluorescence spectroscopy is a powerful method for the investigation of protein dynamics. However, the time resolution of single-molecule fluorescence spectroscopy is usually limited to a few milliseconds. To improve the time resolution, we developed the line-confocal microscope combined with microfluidic chip. By using the device, we could trace the time evolution of FRET efficiency from single molecules with the time resolution of about 100 μs for the observation time of 5 ms. As the first example of the developed method, we investigated the equilibrium unfolding transitions of two mutants of the B domain of protein A (BdpA) doubly labeled with donor and acceptor fluorophores. In the case for the mutant with a shorter donor-acceptor distance, the FRET traces revealed a conformational heterogeneity in the unfolded state. In the case for the mutant with a longer donor-acceptor distance, the presence of a minor conformation in the native state was detected. These results demonstrate that the developed method can reveal the complexity in the apparent two-state folding of BdpA. To track the conformational fluctuations of denatured proteins and the fast protein folding transition, we are improving the system. To obtain the better time resolution without sacrificing the observation time, we built a new system based on hybrid photo detectors (HPD). HPD is a photon-counting detector which achieves a high sensitivity and a large effective area. By introducing HPD to the line-confocal microscope, we could obtain the single-molecule FRET traces with the time resolution of 10 μs and the observation time of more than 10 ms. The traces of BdpA obtained by using the new system exhibited the submillisecond dynamics in the unfolded state, the small fluctuations in the native state and the folding transitions.