Intramolecular Fluorescent Protein Association in a Class of Zinc FRET Sensors Leads to Increased Dynamic Range

Published: April 03, 2019

Author(s)

Joshua D. Slocum, Amy E. Palmer, Ralph Jimenez

Abstract

Genetically encoded Förster resonance energy transfer (FRET) sensors enable the visualization of biological ions, molecules, and processes in live cells. However, despite their widespread use, the molecular states that determine sensor performance are usually poorly understood, which limits efforts to improve them. We used dynamic light scattering and time resolved fluorescence anisotropy to gain insight into the sensing mechanism of ZifCV1.173, a Zn2+ FRET sensor. We found that the dynamic range (DR) of ZifCV1.173 was dominated by the FRET efficiency of the Zn2+-free state, which was compact with the donor and acceptor fluorescent proteins closely interacting. Mutating the donor-acceptor interface revealed that the DR of ZifCV1.173 could be increased or decreased by promoting or disrupting the donor-acceptor interaction, respectively. Adapting the same mutations to a related sensor showed the same pattern of DR tuning, supporting our proposed sensing mechanism and suggesting that intramolecular donor-acceptor interactions could be exploited to increase the DR of other FRET sensors.
Citation: Journal of Physical Chemistry
Volume: 123
Pub Type: Journals

Keywords

Anisotropy, biosensor, dynamic light scattering, fluorescent protein
Created April 03, 2019, Updated June 20, 2019