Protein aggregation involves the assembly of partially misfolded proteins into oligomeric and higher‐order structures that have been associated with several neurodegenerative diseases. However, numerous questions relating to protein aggregation remain unanswered due to the lack of available tools for visualization of these species in living cells. We recently developed a fluorogenic method named aggregation tag (AggTag), and presented the AggTag probe P1, based on a Halo‐tag ligand, to report on the aggregation of a protein of interest (POI) in live cells. However, the Halo‐tag‐based AggTag method only detects the aggregation of one specific POI at a time. In this study, we have expanded the AggTag method by using SNAP‐tag technology to enable fluorogenic and biorthogonal detection of the aggregation of two different POIs simultaneously in live cells. A new AggTag probe—P2, based on a SNAP‐tag ligand bearing a green solvatochromic fluorophore—was synthesized for this purpose. Using confocal imaging and chemical crosslinking experiments, we confirmed that P2 can also report both on soluble oligomers and on insoluble aggregates of a POI fused with SNAP‐tag in live cells. Ultimately, we showed that the orthogonal fluorescence of P1 and P2 allows for simultaneous visualization of two different pathogenic protein aggregates in the same cell.
A peptidyl fluorescent chemosensor for heparin was synthesized by conjugating a pyrene fluorophore with the heparin-binding peptide. The fluorescent chemosensor (Py12; pyrene-RKRLQVQLSIRT) showed a highly sensitive ratiometric response to nanomolar concentrations of heparin in aqueous solutions at physiological pH by increasing excimer emission intensity at 500 nm with a concomitant decrease in monomer emission intensity at 400 nm. Py12 showed a sensitive ratiometric response to heparin over a wide pH range (1.5 ≤ pH ≤ 11.5) and exhibited high selectivity for heparin compared to other biological competitors, such as hyaluronic acid and chondroitin sulfate. Py12 sensitively and ratiometrically detected nanomolar concentrations of heparin in biologically relevant samples containing human serum and human plasma, respectively. The detection limit of Py12 was 34 pM (R(2) = 0.997) for heparin in an aqueous buffer solutions containing 5% human serum and 33 pM (R(2) = 0.994) for heparin in aqueous buffer solutions containing 5% human plasma. Py12 had sufficient sensitivity and selectivity for ratiometrically detecting a nanomolar concentration of heparin, indicating that the peptide-base chemosensor provides a potential tool for monitoring heparin levels in clinical plasma samples.
While organic donor‐acceptor (D‐A) molecules are widely employed in multiple areas, the application of more D‐A molecules could be limited because of an inherent polarity sensitivity that inhibits photochemical processes. Presented here is a facile chemical modification to attenuate solvent‐dependent mechanisms of excited‐state quenching through addition of a β‐carbonyl‐based polar substituent. The results reveal a mechanism wherein the β‐carbonyl substituent creates a structural buffer between the donor and the surrounding solvent. Through computational and experimental analyses, it is demonstrated that the β‐carbonyl simultaneously attenuates two distinct solvent‐dependent quenching mechanisms. Using the β‐carbonyl substituent, improvements in the photophysical properties of commonly used D‐A fluorophores and their enhanced performance in biological imaging are shown.
Aberrant protein aggregation leads to various human diseases, but little is known about the physical chemical properties of these aggregated proteins in cells. Herein, we developed a boron-dipyrromethene (BODIPY)-based HaloTag probe, whose conjugation to HaloTag-fused proteins allows us to study protein aggregates using both fluorescence intensity and lifetime. Modulation of BODIPY fluorophore reveals key structural features to attain the dual function. The optimized probe exhibits increased fluorescence intensity and elongated fluorescence lifetime in protein aggregates. Fluorescence lifetime imaging using this probe indicates that protein aggregates afford different viscosity in the forms of soluble oligomers and insoluble aggregates in live cells. The strategy presented in this work can be extended to enable a wide class of HaloTag probes that can be used to study a variety of physical properties of protein aggregates, thus helping unravel the pathogenic mechanism and develop therapeutic strategy.
A peptide-based ensemble for the detection of cyanide ions in 100% aqueous solutions was designed on the basis of the copper binding motif. 7-Nitro-2,1,3-benzoxadiazole-labeled tripeptide (NBD-SSH, NBD-SerSerHis) formed the ensemble with Cu(2+), leading to a change in the color of the solution from yellow to orange and a complete decrease of fluorescence emission. The ensemble (NBD-SSH-Cu(2+)) sensitively and selectively detected a low concentration of cyanide ions in 100% aqueous solutions by a colorimetric change as well as a fluorescent change. The addition of cyanide ions instantly removed Cu(2+) from the ensemble (NBD-SSH-Cu(2+)) in 100% aqueous solutions, resulting in a color change of the solution from orange to yellow and a "turn-on" fluorescent response. The detection limits for cyanide ions were lower than the maximum allowable level of cyanide ions in drinking water set by the World Health Organization. The peptide-based ensemble system is expected to be a potential and practical way for the detection of submicromolar concentrations of cyanide ions in 100% aqueous solutions.
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