Abstract
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.
Highlights
During the past decades, fluorescent proteins (FPs) have been widely used as tools in fluorescence microscopy to address a broad array of biological and biomedical questions
Following the strategy outlined in the work by Eshaghi et al (2015), we started by creating two chimeric proteins in which rsGreen1 and rsGreenF were directly fused to the N-terminus of the Enhancer nanobody
We set out to determine whether the direct fusion of the Enhancer nanobody to the rsGreen series of photochromic fluorescent proteins resulted in similar advantages as described previously for non-photochromic FPs [23,29]
Summary
The discovery and development of photoresponsive FPs has been of particular interest [1,4] This class of “smart” fluorescent proteins possesses controllable dynamic fluorescence emission, which enables their use for several sub-diffraction imaging techniques such as (fluorescence) photoactivated localization microscopy ((f)PALM) [5,6], reversible saturable optical linear fluorescence transitions (RESOLFT) [7,8,9], non-linear structured illumination microscopy (NL-SIM) [10,11], and photochromic stochastic optical fluctuation imaging (pcSOFI) [12,13]. Plenty of effort has been devoted to creating more optimized photoresponsive FPs, as well as to achieving a fundamental understanding of the mechanisms through which this “smart” behavior is defined [15,16,17,18,19,20,21]
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