Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

Abstract

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Actin visualization at a glance

“…For instance, a recent publication has demonstrated that the subset of F-actin that is recognized by diverse ABDs can be altered by the fluorescent protein or the linker sequence between the ABD and the fluorescent protein (Lemieux et al, 2014). New applications for fluorescent labeling of proteins have become available recently, such as the use of Y-FAST, a small monomeric protein tag enabling reversible binding and activation of a cell-permeant and nontoxic fluorogenic molecule (Plamont et al, 2016), or the flavoprotein improved LOV (iLOV), a fluorescent protein based on the light, oxygen or voltage domain (LOV) from a variety of sources (Buckley et al, 2015). Both molecules offer advantages over GFP and other related fluorescent reporters owing to their relatively small size.…”

Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore

“…Customized fluorescent proteins can be generated by engineering naturally occurring scaffolds to bind synthetic chromophores (Paige et al, 2011; Plamont et al, 2016; Tamura and Hamachi, 2014; Yapici et al, 2015). Chromophore-dependent microbial opsins provide an excellent platform for this approach since modified retinals are well accepted and their incorporation can dramatically alter and enhance opsin properties (Albeck et al, 1989; Asato et al, 1990; AzimiHashemi et al, 2014; Gaertner et al, 1981; Ganapathy et al, 2015; Hoischen et al, 1997; Nielsen, 2009; Sineshchekov et al, 2012).…”

“…In order to access Arch variants with bright NIR emission, we drew inspiration from previous demonstrations that desirable fluorescent protein properties could be obtained by expanding the limited repertoire of naturally known chromophores (Plamont et al, 2016; Tamura and Hamachi, 2014; Yapici et al, 2015). Spectral properties of the natural ATR chromophore (Figure 1 A , Compound 1) can be modulated by adding electron-withdrawing groups (Gaertner et al, 1981; Hendrickx et al, 1995) or changing the length of the conjugated π-bond system (Albeck et al, 1989; Nielsen, 2009).…”

Fluorogenic Protein‐Based Strategies for Detection, Actuation, and Sensing

“…FAP systems based on single‐chain antibodies are relatively large, resulting in fluorescent complexes on the order of GFP or larger. A smaller alternative non‐covalent system is the Fluoresence‐activating and Absorption‐Shifiting Tag (FAST), which was recently developed by directed evolution of the bacterial photoreceptor photoactive yellow protein (PYP) . FAST reversibly and quickly binds a family of hydroxybenzylidene rhodanine derivatives to generate complexes with emission wavelengths from green‐yellow to orange‐red .…”

“…A smaller alternative non‐covalent system is the Fluoresence‐activating and Absorption‐Shifiting Tag (FAST), which was recently developed by directed evolution of the bacterial photoreceptor photoactive yellow protein (PYP) . FAST reversibly and quickly binds a family of hydroxybenzylidene rhodanine derivatives to generate complexes with emission wavelengths from green‐yellow to orange‐red . Furthermore, it has been shown to be an effective fluorescence marker in bacteria, yeast, mammalian cell culture, and zebrafish.…”