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Long-term observation of fluorescence of free single molecules to explore protein-folding energy landscapes.

Journal of the American Chemical Society (2012-06-14)
Kiyoto Kamagata, Toshifumi Kawaguchi, Yoshitomo Iwahashi, Akinori Baba, Kazuya Fujimoto, Tamiki Komatsuzaki, Yoshihiro Sambongi, Yuji Goto, Satoshi Takahashi
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 532 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
Sigma-Aldrich
Atto 532, BioReagent, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 532 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)

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