Quantitative interrogation of protein co-aggregation using multi-color fluorogenic protein aggregation sensors

Abstract

Co‐aggregation of multiple pathogenic proteins is common in neurodegenerative diseases but deconvolution of such biochemical process is challenging. Herein, we developed a dual‐color fluorogenic thermal shift assay to simultaneously report...

A study of protein–drug interaction based on solvent-induced protein aggregation by fluorescence correlation spectroscopy

“…29 Wild-type full-length DHFR with or without EGFP tags used in our experiments was expressed and purified from E. coli cells as described in the ref. 30. The purified proteins were identified by SDS-PAGE and stained with Coomassie Brilliant Blue (shown in Fig.…”

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.…”

Deciphering the Structure and Formation of Amyloids in Neurodegenerative Diseases With Chemical Biology Tools

“…The directed evolution of SrtA to recognize the LMVGG sequence of Aβ (residues 34–38) enabled labeling of endogenous Aβ in human CSF for sensitive detection and conjugating a hydrophilic peptide to Aβ42 that blocks amyloid aggregation ( Podracky et al, 2021 ). Dual-color fluorogenic aggregation sensors were introduced by SML to monitor protein co-aggregation in transthyretin amyloidosis by a thermal shift assay ( Bai et al, 2021 ).…”