Quantitative interrogation of protein co-aggregation using multi-color fluorogenic protein aggregation sensors |
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Authors: | Yulong Bai Wang Wan Yanan Huang Wenhan Jin Haochen Lyu Qiuxuan Xia Xuepeng Dong Zhenming Gao Yu Liu |
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Institution: | CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023 China.; University of Chinese Academy of Sciences, Beijing 100049 China ; The Second Hospital of Dalian Medical University, 467 Zhongshan Road, Dalian 116044 China |
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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 on the aggregation of two different proteins and quantitatively study their thermodynamic stability during co-aggregation. Expansion of spectral coverage was first achieved by developing multi-color fluorogenic protein aggregation sensors. Orthogonal detection was enabled by conjugating sensors of minimal fluorescence crosstalk to two different proteins via sortase-tag technology. Using this assay, we quantified shifts in melting temperatures in a heterozygous model protein system, revealing that the thermodynamic stability of wild-type proteins was significantly compromised by the mutant ones but not vice versa. We also examined how small molecule ligands selectively and differentially interfere with such interplay. Finally, we demonstrated these sensors are suited to visualize how different proteins exert influence on each other upon their co-aggregation in live cells.A little leak will sink a great ship! We prepared a series of multi-color protein aggregation sensors and developed a dual-color thermal shift assay to simultaneously and quantitatively report on protein co-aggregation of two different proteins. |
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