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

Isabella R Walter1, Baylee A Smith1, Dominic Castanzo2,3

  • 1Department of Chemistry & Biochemistry, University of Toledo, Toledo, OH 43606.

Biorxiv : the Preprint Server for Biology
|January 27, 2025
PubMed

Insights

Researchers developed a new method using the fluorescent protein mEOS to study AAA+ unfoldase activity in membrane proteins. This approach overcomes previous limitations, enabling functional analysis of these crucial cellular machines.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Biology

Background:

  • The fluorescent protein mEOS serves as a reporter for AAA+ (ATPases Associated with diverse cellular Activities) unfoldase activity.
  • Many AAA+ proteins are involved in processing membrane proteins, making them important targets for study.
  • Previous attempts to fuse mEOS directly to membrane proteins resulted in loss of fluorescence, hindering research.

Purpose of the Study:

  • To adapt the mEOS reporter system for studying AAA+ unfoldase activity specifically with membrane protein substrates.
  • To overcome the fluorescence quenching issue encountered with direct genetic fusion of mEOS to membrane proteins.

Main Methods:

  • Purification of mEOS and a AAA+ degron as separate components.
  • Covalent linkage of purified mEOS and the AAA+ degron using Sortase enzyme.
  • Photoconversion of the linked mEOS-degron construct for functional analysis.

Main Results:

  • The novel Sortase-mediated linkage strategy successfully preserved mEOS fluorescence when associated with a AAA+ degron.
  • This method enables the functional analysis of AAA+ proteins interacting with membrane proteins, overcoming previous limitations.

Conclusions:

  • A broadly applicable platform has been developed for studying membrane-associated AAA+ proteins.
  • This innovative approach enhances the utility of mEOS as a reporter for AAA+ unfoldase activity in complex membrane protein systems.
Keywords:
AAA+ proteinfluorescent proteinmembrane proteinprotein engineeringproteostasis

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