Photodegradable by Yellow-Orange Light degFusionRed Optogenetic Module with Autocatalytically Formed Chromophore

Abstract

Optogenetic systems driven by yellow-orange light are required for the simultaneous regulation of several cellular processes. We have engineered the red fluorescent protein FusionRed into a 26 kDa monomeric optogenetic module, called degFusionRed. Unlike other fluorescent protein-based optogenetic domains, which exhibit light-induced self-inactivation by generating reactive oxygen species, degFusionRed undergoes proteasomal degradation upon illumination with 567 nm light. Similarly to the parent protein, degFusionRed has minimal absorbance at 450 nm and above 650 nm, making it spectrally compatible with blue and near-infrared-light-controlled optogenetic tools. The autocatalytically formed chromophore provides degFusionRed with an additional advantage over most optogenetic tools that require the binding of the exogenous chromophores, the amount of which varies in different cells. The degFusionRed efficiently performed in the engineered light-controlled transcription factor and in the targeted photodegradation of the protein of interest, demonstrating its versatility as the optogenetic module of choice for spectral multiplexed interrogation of various cellular processes.

Influence of fluorescence lifetime selections and conformational flexibility on brightness of FusionRed variants

“…[16][17][18][19] We recently combined lifetime-based selections with rational mutagenesis to develop brighter FusionRed (FR) variants, notably FR-MQV (FusionRed L175M, M42Q, C159V; =0.53; =2.8 ns), which is the brightest FR variant to date. 20 FR and its progeny are known for their high fidelity cellular localization and low toxicity, [21][22][23] utility in live-cell imaging, and gene expression studies. [21][22][23][24] Furthermore, the fluorescence blinking of FR variants at the single-molecule level has been utilized in super-resolution microscopy.…”

“…20 FR and its progeny are known for their high fidelity cellular localization and low toxicity, [21][22][23] utility in live-cell imaging, and gene expression studies. [21][22][23][24] Furthermore, the fluorescence blinking of FR variants at the single-molecule level has been utilized in super-resolution microscopy. [25][26][27] In the course of developing FR-MQV, we identified the M42Q mutation, which resulted in a significant increase (by ~27%) in quantum yield and lifetime (by ~20%).…”

Influence of Fluorescence Lifetime Selections and Conformational Flexibility on Brightness of FusionRed Variants

“…We recently combined lifetime-based selections with rational mutagenesis to develop brighter FusionRed (FR) variants, notably FR-MQV (FusionRed L175M, M42Q, C159V; ϕ = 0.53; τ = 2.8 ns), which is the brightest FR variant to date . FR and its progeny are known for their high fidelity cellular localization and low toxicity, − utility in live-cell imaging, and gene expression studies. − Furthermore, the fluorescence blinking of FR variants at the single-molecule level has been utilized in super-resolution microscopy. − In the course of developing FR-MQV, we identified the M42Q mutation, which resulted in a significant increase (by ∼27%) in quantum yield and lifetime (by ∼20%). The inspiration to site-saturate this position in the sequence stemmed from our ultrafast spectroscopy studies on hydrogen bonding of the acylimine end of the chromophore in closely related FPs .…”