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Issue 3, 2013
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Single-molecule photon stamping FRET spectroscopy study of enzymatic conformational dynamics

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Abstract

The fluorescence resonant energy transfer (FRET) from a donor to an acceptor via transition dipole–dipole interactions decreases the donor's fluorescent lifetime. The donor's fluorescent lifetime decreases as the FRET efficiency increases, following the equation: EFRET = 1 – τDA/τD, where τD and τDA are the donor fluorescence lifetime without FRET and with FRET. Accordingly, the FRET time trajectories associated with single-molecule conformational dynamics can be recorded by measuring the donor's lifetime fluctuations. In this article, we report our work on the use of a Cy3/Cy5-labeled enzyme, HPPK to demonstrate probing single-molecule conformational dynamics in an enzymatic reaction by measuring single-molecule FRET donor lifetime time trajectories. Compared with single-molecule fluorescence intensity-based FRET measurements, single-molecule lifetime-based FRET measurements are independent of fluorescence intensity. The latter has an advantage in terms of eliminating the analysis background noise from the acceptor fluorescence detection leak through noise, excitation light intensity noise, or light scattering noise due to local environmental factors, for example, in a AFM-tip correlated single-molecule FRET measurements. Furthermore, lifetime-based FRET also supports simultaneous single-molecule fluorescence anisotropy.

Graphical abstract: Single-molecule photon stamping FRET spectroscopy study of enzymatic conformational dynamics

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Publication details

The article was received on 21 Aug 2012, accepted on 19 Sep 2012 and first published on 24 Sep 2012


Article type: Paper
DOI: 10.1039/C2CP42944F
Citation: Phys. Chem. Chem. Phys., 2013,15, 770-775
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    Single-molecule photon stamping FRET spectroscopy study of enzymatic conformational dynamics

    Y. He, M. Lu and H. P. Lu, Phys. Chem. Chem. Phys., 2013, 15, 770
    DOI: 10.1039/C2CP42944F

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