Hetero-FRET Sensors in Vitro (2021-03-26)

Abstract

Living cells are crowded with macromolecules and organelles, which are believed toinfluence a wide range of biochemical processes and pathological conditions of eukaryoticcells. A deeper understanding of this correlation can lead to breakthroughs in findingtreatments to diseases. Förster resonance energy transfer (FRET) is considered as amolecular ruler that is able to quantify protein-protein interactions and structuralconformation in a range of biomolecules in both controlled environments and in livingcells. Currently, a series of genetically encoded fluorescent protein constructs have beendesigned to quantify both the macromolecular crowding and ionic strength using FRET. Itis capable of transferring energy non-radiatively from an excited donor to an acceptor. Thistransfer is done without the donor emitting a photon and the acceptor absorbing a photon.Researchers have seen how increasingly beneficial FRET is when it comes to accuratespatial measurements and detecting biological organizations. The focus is to examine theeffects of the differing donor (mCerulean3 and mTurquoise2.1) on the biosensors’sensitivity to either macromolecular crowding or ionic strength with mCitrine acting as theFRET acceptor. These FRET pairs are connected via a flexible hinge that is designed toreport changes in the environment surrounding the biosensor. In one family of FRETsensors, the efficiency of energy transfer increases as macromolecular crowding increases;that is, the donor and acceptor come in close proximity. In another family of FRET sensors,energy transfer decreases due to electrostatic screening between charged ?-helices in thehinge, thereby leading to an increase in the donor-acceptor distance between the donorand acceptor. There are many analysis tools used to calculate the FRET efficiency of theFRET probes for either macromolecular crowding or ionic strength. The analysis tools wespecifically used are fluorescence lifetime measurements, fluorescence correlationspectroscopy, and time-resolved polarized fluorescence anisotropy. We compared theenergy transfer efficiencies of the donors, mCerulean3 and mTurquoise2.1 with themCitrine acceptor. These results will inform future design of engineered protein-basedbiosensors.