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Int. J. Mol. Sci. 2017, 18(9), 2015; doi:10.3390/ijms18092015

Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

Laboratory for Nanobiology, Department of Chemistry, KU Leuven, Celestijnenlaan 200G, 3001 Leuven, Belgium
These authors contributed equally to this work.
Author to whom correspondence should be addressed.
Received: 16 August 2017 / Revised: 8 September 2017 / Accepted: 11 September 2017 / Published: 20 September 2017
(This article belongs to the Special Issue Fluorescent Proteins)
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Reversibly switchable fluorescent proteins (RSFPs) enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties. View Full-Text
Keywords: fluorescent proteins; reversible photoswitching; photochromism; switching fatigue; contrast; nanobody; rsGreen; spectroscopy fluorescent proteins; reversible photoswitching; photochromism; switching fatigue; contrast; nanobody; rsGreen; spectroscopy

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Roebroek, T.; Duwé, S.; Vandenberg, W.; Dedecker, P. Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization. Int. J. Mol. Sci. 2017, 18, 2015.

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