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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 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.

SUBMITTER: Bai Y

PROVIDER: S-EPMC8221170 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nucleic acid-based electrochemical sensors (NBEs) can support continuous and highly selective molecular monitoring in biological fluids, both in vitro and in vivo, via affinity-based interactions. Such interactions afford a sensing versatility that is not supported by strategies that depend on target-specific reactivity. Thus, NBEs have significantly expanded the scope of molecules that can be monitored continuously in biological systems. However, the technology is limited by the lability of the thiol-based monolayers employed for sensor fabrication. Seeking to understand the main drivers of monolayer degradation, we studied four possible mechanisms of NBE decay: (i) passive desorption of monolayer elements in undisturbed sensors, (ii) voltage-induced desorption under continuous voltammetric interrogation, (iii) competitive displacement by thiolated molecules naturally present in biofluids like serum, and (iv) protein binding. Our results indicate that voltage-induced desorption of monolayer elements is the main mechanism by which NBEs decay in phosphate-buffered saline. This degradation can be overcome by using a voltage window contained between -0.2 and 0.2 V vs Ag|AgCl, reported for the first time in this work, where electrochemical oxygen reduction and surface gold oxidation cannot occur. This result underscores the need for chemically stable redox reporters with more positive reduction potentials than the benchmark methylene blue and the ability to cycle thousands of times between redox states to support continuous sensing for long periods. Additionally, in biofluids, the rate of sensor decay is further accelerated by the presence of thiolated small molecules like cysteine and glutathione, which can competitively displace monolayer elements even in the absence of voltage-induced damage. We hope that this work will serve as a framework to inspire future development of novel sensor interfaces aiming to eliminate the mechanisms of signal decay in NBEs.

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

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