A SNAP-tag fluorogenic probe mimicking the chromophore of the red fluorescent protein Kaede.

Self-labelling protein tags with fluorogenic probes serve as great fluorescence imaging tools to understand key questions of protein dynamics and functions in living cells. In the present study, we report a SNAP-tag fluorogenic probe 4c mimicking the chromophore of the red fluorescent protein Kaede. The molecular rotor properties of 4c were utilized as a fluorogenic probe for SNAP-tag, such that conjugation with SNAPf protein leads to inhibition of twisted intramolecular charge transfer, triggering fluorogenecity. Upon conjugation with SNAPf, 4c exhibited approximately a 90-fold enhancement in fluorescence intensity with fast labelling kinetics (k2 = 15 000 M-1 s-1). Biochemical and spectroscopic studies confirmed that fluorescence requires formation of folded SNAPf·4c covalent conjugate between Cys 145 and 4c. Confocal microscopy and flow cytometry showed that 4c is capable of detecting SNAPf proteins or SNAPf fused with a protein of interest in living cells. This work provides a framework to develop the large family of FP chromophores into fluorogenic probes for self-labelling protein tags.

A HaloTag-Based Multicolor Fluorogenic Sensor Visualizes and Quantifies Proteome Stress in Live Cells Using Solvatochromic and Molecular Rotor-Based Fluorophores.

Protein homeostasis, or proteostasis, is essential for cellular fitness and viability. Many environmental factors compromise proteostasis, induce global proteome stress, and cause diseases. The proteome stress sensor is a powerful tool for dissecting the mechanism of cellular stress and finding therapeutics that ameliorate these diseases. In this work, we present a multicolor HaloTag-based sensor (named AgHalo) to visualize and quantify proteome stresses in live cells. The current AgHalo sensor is equipped with three fluorogenic probes that turn on fluorescence when the sensor forms either soluble oligomers or insoluble aggregates upon exposure to stress conditions, both in vitro and in cellulo. In addition, AgHalo probes can be combined with commercially available always-fluorescent HaloTag ligands to enable two-color imaging, allowing for direct visualization of the AgHalo sensor both before and after cells are subjected to stress conditions. Finally, pulse-chase experiments can be performed to discern changes in the cellular proteome in live cells by first forming the AgHalo conjugate and then either applying or removing stress at any desired time point. In summary, the AgHalo sensor can be used to visualize and quantify proteome stress in live cells, a task that is difficult to accomplish using previous always-fluorescent methods. This sensor should be suited to evaluating cellular proteostasis under various exogenous stresses, including chemical toxins, drugs, and environmental factors.

Modulation of Fluorescent Protein Chromophores To Detect Protein Aggregation with Turn-On Fluorescence.

We present a fluorogenic method to visualize misfolding and aggregation of a specific protein-of-interest in live cells using structurally modulated fluorescent protein chromophores. Combining photophysical analysis, X-ray crystallography, and theoretical calculation, we show that fluorescence is triggered by inhibition of twisted-intramolecular charge transfer of these fluorophores in the rigid microenvironment of viscous solvent or protein aggregates. Bioorthogonal conjugation of the fluorophore to Halo-tag fused protein-of-interests allows for fluorogenic detection of both misfolded and aggregated species in live cells. Unlike other methods, our method is capable of detecting previously invisible misfolded soluble proteins. This work provides the first application of fluorescent protein chromophores to detect protein conformational collapse in live cells.

A Molecular Rotor-Based Halo-Tag Ligand Enables a Fluorogenic Proteome Stress Sensor to Detect Protein Misfolding in Mildly Stressed Proteome.

Cellular stress leads to disruption of protein homeostasis (proteostasis) that is associated with global misfolding and aggregation of the endogenous proteome. Monitoring stress-induced proteostasis deficiency remains one of the major technical challenges facing established sensors of this process. Available sensors use solvatochromic fluorophores to detect protein aggregation in forms of soluble oligomers or insoluble aggregates when cells are subjected to severe stress conditions. Misfolded monomers induced by mild stresses, however, remain largely invisible to these sensors. Here, we describe a fluorogenic proteome stress sensor by conjugating a fluorescent molecular rotor with a metastable Halo-tag protein domain that contains a K73T mutation (named AgHalo hereinafter). In nonstressed cells, the AaHalo sensor remains largely folded and the AgHalo•ligand conjugate is fluorescent dark in the folded state. Under various stress conditions, the AgHalo sensor has been established to form both soluble and insoluble aggregates along with metastable proteins of the endogenous cellular proteome. Thus, the AgHalo•ligand conjugate fluoresces strongly when the sensor forms misfolded monomers (a 16-fold increase) or aggregates in both soluble and insoluble forms (a 20-fold increase). Compared to the solvatochromic fluorophore-based sensor, we demonstrate that the molecular rotor-based sensor not only is more effective in detecting mild proteome stress that induces primarily misfolding conformations, but also exhibits a higher fluorescence signal in detecting more severe proteome stress that involves protein aggregates. Thus, the conjugation of a fluorescent molecular rotor to AgHalo further improves the capacity of this sensor to detect conditions of proteome stress. This work highlights the utility of molecular rotor-based fluorophores in direct visualization of the protein aggregation cascade in live cells, providing new methodologies for real-time analyses of cellular proteostasis upon exposure to different types of stress conditions.

The Cation-π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification.

The design of fluorogenic probes for a Halo tag is highly desirable but challenging. Previous work achieved this goal by controlling the chemical switch of spirolactones upon the covalent conjugation between the Halo tag and probes or by incorporating a "channel dye" into the substrate binding tunnel of the Halo tag. In this work, we have developed a novel class of Halo-tag fluorogenic probes that are derived from solvatochromic fluorophores. The optimal probe, harboring a benzothiadiazole scaffold, exhibits a 1000-fold fluorescence enhancement upon reaction with the Halo tag. Structural, computational, and biochemical studies reveal that the benzene ring of a tryptophan residue engages in a cation-π interaction with the dimethylamino electron-donating group of the benzothiadiazole fluorophore in its excited state. We further demonstrate using noncanonical fluorinated tryptophan that the cation-π interaction directly contributes to the fluorogenicity of the benzothiadiazole fluorophore. Mechanistically, this interaction could contribute to the fluorogenicity by promoting the excited-state charge separation and inhibiting the twisting motion of the dimethylamino group, both leading to an enhanced fluorogenicity. Finally, we demonstrate the utility of the probe in no-wash direct imaging of Halo-tagged proteins in live cells. In addition, the fluorogenic nature of the probe enables a gel-free quantification of fusion proteins expressed in mammalian cells, an application that was not possible with previously nonfluorogenic Halo-tag probes. The unique mechanism revealed by this work suggests that incorporation of an excited-state cation-π interaction could be a feasible strategy for enhancing the optical performance of fluorophores and fluorogenic sensors.

AgHalo: A Facile Fluorogenic Sensor to Detect Drug-Induced Proteome Stress.

Drug-induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug-induced proteome stress prior to cell death. An aggregation prone Halo-tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins.

Stress-induced protein disaggregation in the endoplasmic reticulum catalysed by BiP.

Protein synthesis is supported by cellular machineries that ensure polypeptides fold to their native conformation, whilst eliminating misfolded, aggregation prone species. Protein aggregation underlies pathologies including neurodegeneration. Aggregates' formation is antagonised by molecular chaperones, with cytoplasmic machinery resolving insoluble protein aggregates. However, it is unknown whether an analogous disaggregation system exists in the Endoplasmic Reticulum (ER) where ~30% of the proteome is synthesised. Here we show that the ER of a variety of mammalian cell types, including neurons, is endowed with the capability to resolve protein aggregates under stress. Utilising a purpose-developed protein aggregation probing system with a sub-organellar resolution, we observe steady-state aggregate accumulation in the ER. Pharmacological induction of ER stress does not augment aggregates, but rather stimulate their clearance within hours. We show that this dissagregation activity is catalysed by the stress-responsive ER molecular chaperone - BiP. This work reveals a hitherto unknow, non-redundant strand of the proteostasis-restorative ER stress response.

Quantification of Cellular Proteostasis in Live Cells by Fluorogenic Assay Using the AgHalo Sensor.

Proper cellular proteostasis is essential to cellular fitness and viability. Exogenous stress conditions compromise proteostasis and cause aggregation of cellular proteins. We have developed a fluorogenic sensor (AgHalo) to quantify stress-induced proteostasis deficiency. The AgHalo sensor uses a destabilized HaloTag variant to represent aggregation-prone cellular proteins and is equipped with a series of fluorogenic probes that exhibit a fluorescence increase when the sensor forms either soluble oligomers or insoluble aggregates. Herein, we present protocols that describe how the AgHalo sensor can be employed to visualize and quantify proteome stress in live cells using a direct fluorescence read-out and visualization with a fluorescence microplate reader and a microscope. Additionally, protocols for using the AgHalo sensor in combination with fluorogenic probes and commercially available HaloTag probes to enable two-color imaging experiments are described. These protocols will enable use of the AgHalo sensor to visualize and quantify proteostasis in live cells, a task that is difficult to accomplish using previous, always-fluorescent methods. © 2018 by John Wiley & Sons, Inc.

Monitoring Proteome Stress in Live Cells Using HaloTag-Based Fluorogenic Sensor.

Fluorescent folding sensor is a powerful tool to detect proteome stresses, including heat, osmotic, oxidative, and drug induced stresses. Monitoring proteome stress using these sensors allows us to dissect the mechanism of cellular stress and find therapeutics that ameliorate stress related diseases. Here we present a HaloTag-based fluorogenic proteome stress sensor (AgHalo) to robustly detect and quantify proteome stresses in live cells. We describe how proteome stresses are monitored in both bacterial and mammalian live cells using fluorescence confocal microscope and fluorescence plate reader.