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bioRxiv · 10.1101/2025.01.14.633048

Overcoming Fluorescence Loss in mEOS-based AAA+ Unfoldase Reporters Through Covalent Linkage

Walter, I.·Smith, B.·Castanzo, D.·Wohlever, M. L.
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Abstract

Recent work has demonstrated that the soluble photoconvertable fluorescent protein mEOS can be a reporter for AAA+ (ATPases Associated with diverse cellular Activities) unfoldase activity. Given that many AAA+ proteins process membrane proteins, we sought to adapt mEOS for use with membrane protein substrates. However, direct genetic fusion of mEOS to a membrane protein completely abolished fluorescence, severely limiting the utility of mEOS for studying AAA+ proteins. To circumvent this challenge, we separately purified mEOS and a AAA+ degron, covalently linked them via Sortase, and photoconverted the linked construct. This innovative approach preserves fluorescence and enables functional analysis, offering a broadly applicable platform for the study of membrane associated AAA+ proteins.

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Walter, I., Smith, B., Castanzo, D., Wohlever, M. L.. 2025-01-15. Overcoming Fluorescence Loss in mEOS-based AAA+ Unfoldase Reporters Through Covalent Linkage. https://doi.org/10.1101/2025.01.14.633048

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