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

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.

Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins

“…For example, the difference between the maximal absorption and excitation wavelengths ( ex − abs ) positively correlates with the off-switching quantum yield Q off . This wavelength difference is usually seen as an indicator of ground state heterogeneity within an FP-ensemble, and is consistent with the presence of at least two populations with different photoswitching propensity and brightness in the ground state [21,24]. We will discuss particular insights enabled by these correlations further along in this contribution.…”

“…(D) Zoomed-in region from the second cycle that was used to calculate the dynamic macroscopic properties except for A unrecov and k therm . characterization using a two-level screening assay consisting of transformation in E. coli, in-colony characterization [14], and on-microscope screening in a 96-well format [21]. During the first screening round in bacterial colonies, mutants deemed worthy of further investigation were identified based on their relative brightness, photoswitching kinetics and contrast between the fluorescent and dim state.…”

Reversible photobleaching of photoconvertible SAASoti-FP

“…The photobleaching experiments with different fluorescent proteins exhibit bi-and mono-exponential fluorescence decay dependences [10], [11]. To characterize the photoswitching between 'on' and 'off' forms we recorded the photobleaching ( Fig.…”

“…photobleaching certainly depends on the light exposure intensity. The phenomenon of bi-exponential fluorescence decay was explained by the existence of two protein populations in the excited state that can be reversibly converted to the 'off' state [11]. Previously, this 'multi protein populations' hypothesis was suggested by our group [12] when conducting experiments on KFP and its pH-dependency.…”

Nanobody-Based Probes for Subcellular Protein Identification and Visualization

“…Non-targeted fluorescence can also be reduced by enhancer nanobodies (Roebroek et al, 2015). When the enhancer nanobodies bind with GFP, it increases the fluorescence and stability of the GFP.…”