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

Abstract

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.

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

“…These precip- 4h,S9, and S10). [49] Interestingly,the majority of these probes (A1-A5, A7-A8, B1-B9 in Figure 2a)recovered the fluorescence in crystalline solid state regardless of their substituents (Figures 4g,h, S12, S13, S15, and S16). Some of these probes (A3, A4, B1-B4) do not fluoresce in viscous or nonpolar solvents but exclusively fluoresce in crystalline solid, suggesting that these probes require well-aligned compaction that inhibits all possible bond rotations to turn on the fluorescence.H owever, this stringent requirement cannot be satisfied by viscous solvent (glycerol), nonpolar solvent (dioxane), amorphous solid and protein aggregates that lack of template compact structures.T hese observations may explain why AIEgens were mostly reported as amyloid sensors and sensitization of AIEgens towards nonpolar and viscous environment in this work is necessary for them to detect amorphous protein aggregates of no defined structure.…”

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

“…However, imaging techniques to directly visualize the morphology of protein particles are necessary upon measuring protein liquid-to-liquid phase separation due to its highly dynamic and reversible nature. In addition, we expected that new protein aggregation sensors − ,− ,− and novel fluorogenic chemistries , can potentially assist us in resolving the protein phase separation process in detail. Overall, we present experimental recommendations that researchers may take into consideration when using the turbidity assay to study protein phase separation and further reveal the biochemical mechanism-of-action of these currently incurable protein conformational diseases.…”

“…The working principle of the turbidity assay involves the quantification of the difference in ultraviolet spectra of protein solutions upon phase separation, containing contributions not only from a shift in the spectrum of the tyrosyl group but also from Rayleigh light scattering when there are large differences in the state of molecular aggregation (Figure B). − Turbidity differences can be quantitatively assessed by UV–visible spectrophotometry and dynamic light scattering (DLS) . Such an assay can provide quantitative information to describe the protein phase separation process, including aggregation kinetics from the time-dependent experiment, the severity of aggregation from the optical density end-point reading, melting temperatures from the thermal shift assay, and particle size from dynamic light scattering. − Our laboratories focused on developing multiple types of fluorescent sensors, which aims to monitor and resolve the entire process of protein phase separation and dissect its mechanism-of-action. − Over years, we usually observed reproducibility and accuracy issues when using the turbidity assay and its relevant applications to study protein phase separation. These observations lead to a plausible but previously overlooked concern: the linear range or detection limit issue in the turbidity assay.…”

Regulation of Fluorescence Solvatochromism To Resolve Cellular Polarity upon Protein Aggregation

“…However, it has always been a technical challenge to achieve this goal in live cells because this process involves complex protein conformational transitions. The loss of structural integrity in misfolded and aggregated proteins poses hurdles to rationally develop chemical strategies that selectively target an individual folding state. , In particular, the recent discovery of protein liquid-to-liquid phase-separation (LLPS) behavior of intrinsic disordered proteins further exaggerates such a dilemma. − Thus, chemical probes that can selectively resolve and illuminate different misfolded and aggregated states of protein molecules will advance our understanding of the biochemical basis underlying the protein phase-separation process.…”

“…Toward this goal, efforts have been devoted to the rational design of fluorescent probes to illuminate and differentiate misfolded and aggregated species. , Among them, fluorescent sensors targeting insoluble protein amyloid aggregates have been widely reported, possibly due to the vast availability of amyloid structures revealed by state-of-the-art structural biology methods . The β-sheet compact structure of amyloid aggregates provides defined binding grooves or cavities for fluorescent chemical probes to target selectively .…”