Long-term observation of fluorescence of free single molecules to explore protein-folding energy landscapes.
A method was developed to detect fluorescence intensity signals from single molecules diffusing freely in a capillary cell. A unique optical system based on a spherical mirror was designed to enable quantitative detection of the fluorescence intensity. Furthermore, "flow-and-stop" control of the sample can extend the observation time of single molecules to several seconds, which is more than 1000 times longer than the observation time available using a typical confocal method. We used this method to scrutinize the fluorescence time series of the labeled cytochrome c in the unfolded state. Time series analyses of the trajectories based on local equilibrium state analysis revealed dynamically differing substates on a millisecond time scale. This system presents a new avenue for experimental characterization of the protein-folding energy landscape.
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.
Kiyoto
K
Kamagata
Toshifumi
T
Kawaguchi
Yoshitomo
Y
Iwahashi
Akinori
A
Baba
Kazuya
K
Fujimoto
Tamiki
T
Komatsuzaki
Yoshihiro
Y
Sambongi
Yuji
Y
Goto
Satoshi
S
Takahashi
D005453:Fluorescence; D017510:Protein Folding