Common Pitfalls and Recommendations for Using a Turbidity Assay to Study Protein Phase Separation.

The turbidity assay is commonly exploited to study protein liquid-to-liquid phase separation (LLPS) or liquid-to-solid phase separation (LSPS) processes in biochemical analyses. Herein, we present common pitfalls of this assay caused by exceeding the detection linear range. We showed that aggregated proteins of high concentration and large particle size can lead to inaccurate quantification in multiple applications, including the optical density measurement, the thermal shift assay, and the dynamic light scattering experiment. Finally, we demonstrated that a simple sample dilution of insoluble aggregated protein (LSPS) samples or direct imaging of liquid droplets (LLPS) can address these issues and improve the accuracy of the turbidity assay.

Regulation of Fluorescence Solvatochromism To Resolve Cellular Polarity upon Protein Aggregation.

Common solvatochromic fluorophores exhibit a bathochromic fluorescence emission wavelength shift accompanied by intensity attenuation due to the presence of nonradiative decay pathways at the excited state. Such intrinsic but inevitable fluorescence quenching of solvatochromism impedes its applications to faithfully quantify local polarity, especially in a polar environment. Herein, we report a new donor-π-acceptor (D-π-A) type solvatochromic fluorophore scaffold containing a perfluorophenyl group that exhibits both a solvatochromic emission wavelength shift and a controllable emission intensity upon polarity fluctuation. The regulation of fluorescence solvatochromism and colors was achieved by tuning the aryl donors. We exploited such desired solvatochromism of these probes to monitor protein misfolding and aggregation via wavelength shift. Finally, the polarity of pathogenic aggregated proteins was quantified by HaloTag bioorthogonal labeling technology in live cells. While much effort has been devoted to resolving the morphology of pathogenic aggregated proteins, this work provides quantitative hints regarding the chemical information at this disease-related protein interphase.

A Solvatochromic Fluorescent Probe Reveals Polarity Heterogeneity upon Protein Aggregation in Cells.

We report a crystallization-induced emission fluorophore to quantitatively interrogate the polarity of aggregated proteins. This solvatochromic probe, namely "AggRetina" probe, inherently binds to aggregated proteins and exhibits both a polarity-dependent fluorescence emission wavelength shift and a viscosity-dependent fluorescence intensity increase. Regulation of its polarity sensitivity was achieved by extending the conjugation length. Different proteins bear diverse polarity upon aggregation, leading to different resistance to proteolysis. Polarity primarily decreases during protein misfolding but viscosity mainly increases upon the formation of insoluble aggregates. We quantified the polarity of aggregated protein-of-interest in live cells via HaloTag bioorthogonal labeling, revealing polarity heterogeneity within cellular aggregates. The enriched micro-environment details inside misfolded and aggregated proteins may correlate to their bio-chemical properties and pathogenicity.

Rational Design of Crystallization-Induced-Emission Probes To Detect Amorphous Protein Aggregation in Live Cells.

Unlike amyloid aggregates, amorphous protein aggregates with no defined structures have been challenging to target and detect in a complex cellular milieu. In this study, we rationally designed sensors of amorphous protein aggregation from aggregation-induced-emission probes (AIEgens). Utilizing dicyanoisophorone as a model AIEgen scaffold, we first sensitized the fluorescence of AIEgens to a nonpolar and viscous environment mimicking the interior of amorphous aggregated proteins. We identified a generally applicable moiety (dimethylaminophenylene) for selective binding and fluorescence enhancement. Regulation of the electron-withdrawing groups tuned the emission wavelength while retaining selective detection. Finally, we utilized the optimized probe to systematically image aggregated proteome upon proteostasis network regulation. Overall, we present a rational approach to develop amorphous protein aggregation sensors from AIEgens with controllable sensitivity, spectral coverage, and cellular performance.

Solvatochromic sensors detect proteome aggregation in stressed liver tissues with hepatic cancer and cirrhosis.

Protein misfolding and aggregation involve complex cellular processes with clinical implications in various diseases. However, the detection of aggregated proteomes without defined 3-D structures in a complex biological milieu is challenging. This study utilizes chromone scaffold-based environment-sensitive fluorophores P1 and P2 to detect misfolded and aggregated proteome in stressed liver cells and the liver tissues diseased patients. The reported crystallization induced emission probes (P1 and P2) exhibit both polarity and viscosity sensitivity, with emission intensity and wavelength linearly correlated to viscosity and polarity. Meanwhile, P1 and P2 selectively and generally fluoresce upon binding to various aggregated proteins. In hepatic cells, P2 outperforms P1 in detecting stress-induced global proteome aggregation. In mouse liver tissue upon drug-induced injury, the fluorescence intensity of P2 correlated with the severity of liver injury, serving as an earlier indicator for liver stress prior to ALT/AST increase. The quantification of emission wavelength reveals lower micro-environmental polarity in liver-injury tissue. In patient-derived tissues with hepatic cancer and cirrhosis, P1 and P2 also report on the presence of aggregated proteome. Together, the reported solvatochromic proteome aggregation sensors can detect hepatic proteome aggregation and analyze its local polarity in cultured cell lines, animal model tissues, and human clinical samples.

Derivatizing Nile Red fluorophores to quantify the heterogeneous polarity upon protein aggregation in the cell.

Protein aggregation in the cell is often manifested by the formation of subcellular punctate structures. Herein, we modulated the solvatochromism and solubility of Nile Red fluorophore derivatives to quantitatively study the polarity inside pathogenic protein aggregates, revealing structure- and protein-dependent polarity heterogeneity.

Derivatizing merocyanine dyes to balance their polarity and viscosity sensitivities for protein aggregation detection.

Protein misfolding and aggregation processes involve local polarity and viscosity fluctuation. Herein we modulated the polarity and viscosity sensitivities of merocyanine dyes to detect protein aggregation. We demonstrated how structural modulation balanced these two fluorescence sensitivities and affected the detection of misfolded and aggregated proteins.