Conformational equilibrium analysis of mCerulean3–linker–mCitrine constructs using time-resolved fluorescence measurements in controlled environments

Clint McCue; Sarah A. Mersch; Sarah Bergman; Erin D. Sheets; Ahmed A. Heikal

doi:10.1117/12.2679719

5 October 2023 Conformational equilibrium analysis of mCerulean3–linker–mCitrine constructs using time-resolved fluorescence measurements in controlled environments

Clint McCue, Sarah A. Mersch, Sarah Bergman, Erin D. Sheets, Ahmed A. Heikal

Author Affiliations +

Proceedings Volume 12681, Ultrafast Nonlinear Imaging and Spectroscopy XI; 1268107 (2023) https://doi.org/10.1117/12.2679719
Event: SPIE Optical Engineering + Applications, 2023, San Diego, California, United States

Abstract

Macromolecular crowding and ionic strength in living cells influence a myriad of biochemical processes essential to cell function and survival. For example, macromolecular crowding is known to affect diffusion, biochemical reaction kinetics, protein folding, and protein-protein interactions. In addition, enzymatic activities, protein folding, and cellular osmosis are also sensitive to environmental ionic strength. Recently, genetically encoded mCerulean3-linker-mCitrine constructs have been developed and characterized using time-resolved fluorescence measurements as a function of the amino acid sequence of the linker region as well as the environmental crowding and ionic strength. Here, we investigate the thermodynamic equilibrium of structural conformations of mCerulean3-linker-mCitrine constructs in response to the environmental macromolecular crowding and ionic strength. We have developed a theoretical framework for thermodynamic equilibrium of the structural conformations of these environmental sensors. In addition, we tested these theoretical models for thermodynamic analysis of these donor-linker-acceptor sensors using time-resolved fluorescence measurements as a function of the amino acid sequence of the linker region. Employing ultrafast time-resolved fluorescence measurements for gaining thermodynamic energetics would be helpful for Förster Resonance Energy Transfer (FRET) studies of protein-protein interactions in both living cells and controlled environments.

Conference Presentation

(2023) Published by SPIE. Downloading of the abstract is permitted for personal use only.

Citation Download Citation

Clint McCue, Sarah A. Mersch, Sarah Bergman, Erin D. Sheets, and Ahmed A. Heikal "Conformational equilibrium analysis of mCerulean3–linker–mCitrine constructs using time-resolved fluorescence measurements in controlled environments", Proc. SPIE 12681, Ultrafast Nonlinear Imaging and Spectroscopy XI, 1268107 (5 October 2023); https://doi.org/10.1117/12.2679719

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