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ACS Sens. 2018 Sep 28;3(9):1735-1742. doi: 10.1021/acssensors.8b00473. Epub 2018 Sep 12.

Influence of Fluorescent Protein Maturation on FRET Measurements in Living Cells.

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Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute , University of Groningen , Nijenborgh 4 , 9747 AG Groningen , The Netherlands.
European Research Institute for the Biology of Ageing , University of Groningen , University Medical Center Groningen, 9713 AV Groningen , The Netherlands.
Zernike Institute for Advanced Materials , University of Groningen , Nijenborgh 4 , 9747 AG Groningen , The Netherlands.


Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet it remains necessary to identify and prevent potential artifacts in order to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression in both prokaryotic and eukaryotic cells, which can also be achieved by incorporation of faster-maturing FRET donors. We developed an improved version of the donor mTurquoise2 that matures faster than the parent protein. Our analysis shows that using equal maturing fluorophores in FRET-based sensors or using constitutive low expression conditions helps to reduce maturation-induced artifacts, without the need of additional noise-inducing spectral corrections. In general, we show that monitoring and controlling the maturation of fluorescent proteins in living cells is important and should be addressed in in vivo applications of genetically encoded FRET sensors.


FRET sensors; Förster resonance energy transfer; biosensors; fluorescent protein maturation; macromolecular crowding

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