Integrated fluorescence approach for FRET analysis of environmental sensors

Cody P. Aplin; Taryn M. Kay; Robert C. Miller; Arnold J. Boersma; Erin D. Sheets; Ahmed A. Heikal

doi:10.1117/12.2531656

9 September 2019 Integrated fluorescence approach for FRET analysis of environmental sensors

Cody P. Aplin, Taryn M. Kay , Robert C. Miller , Arnold J. Boersma, Erin D. Sheets, Ahmed A. Heikal

Proceedings Volume 11122, Ultrafast Nonlinear Imaging and Spectroscopy VII; 111220E (2019) https://doi.org/10.1117/12.2531656
Event: SPIE Optical Engineering + Applications, 2019, San Diego, California, United States

Abstract

Förster resonance energy transfer (FRET) is considered as a molecular ruler to quantify protein-protein interactions and structural conformation in a wide range of biomolecules in both controlled environments and in living cells. Here, we have employed integrated fluorescence spectroscopy methods to characterize the energy transfer efficiency and donor-acceptor distance for novel genetically engineered mCerulean3–linker– mCitrine environmental sensors. Based on the amino acids sequences of the linker region, these sensors can be sensitive to either macromolecular crowding or the ionic strength of the surrounding environment. These hetero-FRET sensors also enable us to develop new spectroscopic approaches for quantifying the energy transfer efficiency and the donor-acceptor distance as a means of elucidating the underlying mechanisms for environmental sensing. Ensemble averaging approaches using time-resolved fluorescence and time-resolved fluorescence polarization anisotropy of G12 sensor are highlighted. Our findings in control environments so far are currently being used for complementary studies in living cells.

Conference Presentation

Citation Download Citation

Cody P. Aplin, Taryn M. Kay , Robert C. Miller , Arnold J. Boersma, Erin D. Sheets, and Ahmed A. Heikal "Integrated fluorescence approach for FRET analysis of environmental sensors", Proc. SPIE 11122, Ultrafast Nonlinear Imaging and Spectroscopy VII, 111220E (9 September 2019); https://doi.org/10.1117/12.2531656

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