Kinetics model for the wavelength-dependence of excited-state dynamics of hetero-FRET sensors

Jacob Schwarz; Ryan Leighton; Hannah J. Leopold; Megan Currie; Arnold J. Boersma; Erin D. Sheets; Ahmed A. Heikal

doi:10.1117/12.2274793

7 September 2017 Kinetics model for the wavelength-dependence of excited-state dynamics of hetero-FRET sensors

Jacob Schwarz, Ryan Leighton, Hannah J. Leopold, Megan Currie, Arnold J. Boersma, Erin D. Sheets, Ahmed A. Heikal

Proceedings Volume 10380, Ultrafast Nonlinear Imaging and Spectroscopy V; 103800S (2017) https://doi.org/10.1117/12.2274793
Event: SPIE Optical Engineering + Applications, 2017, San Diego, California, United States

Abstract

Foerster (or fluorescence) resonance energy transfer (FRET) is a powerful tool for investigating protein-protein interactions, in both living cells and in controlled environments. A typical hetero-FRET pair consists of a donor and acceptor tethered together with a linker. The corresponding energy transfer efficiency of a hetero-FRET pair probe depends upon the donor-acceptor distance, relative dipole orientation, and spectral overlap. Because of the sensitivity of the energy transfer efficiency on the donor-acceptor distance, FRET is often referred to as a “molecular ruler”. Time-resolved fluorescence approach for measuring the excited-state lifetime of the donor and acceptor emissions is one of the most reliable approaches for quantitative assessment of the energy transfer efficiency in hetero-FRET pairs. In this contribution, we provide an analytical kinetics model that describes the excited-state depopulation of a FRET probe as a means to predicts the time-resolved fluorescence profile as a function of excitation and detection wavelengths. In addition, we used this developed kinetics model to simulate the time-dependence of the excited-state population of both the donor and acceptor. These results should serve as a guide for our ongoing studies of newly developed hetero-FRET sensors (mCerulean3–linker–mCitrine) that are designed specifically for in vivo studies of macromolecular crowding. The same model is applicable to other FRET pairs with the careful consideration of their steady-state spectroscopy and the experimental design for wavelength- dependence of the fluorescence lifetime measurements.

Citation Download Citation

Jacob Schwarz, Ryan Leighton, Hannah J. Leopold, Megan Currie, Arnold J. Boersma, Erin D. Sheets, and Ahmed A. Heikal "Kinetics model for the wavelength-dependence of excited-state dynamics of hetero-FRET sensors", Proc. SPIE 10380, Ultrafast Nonlinear Imaging and Spectroscopy V, 103800S (7 September 2017); https://doi.org/10.1117/12.2274793

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