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Overcoming fluorescence loss in mEOS-based AAA+ unfoldase reporters through covalent linkage

Isabella R Walter1, Baylee A Smith1, Dominic Castanzo2

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

Protein Expression and Purification
|April 30, 2025
PubMed

Insights

Researchers developed a new method to study membrane protein activity using the fluorescent reporter mEOS. This technique overcomes previous limitations, enabling fluorescence reporting for ATPases Associated with diverse cellular Activities (AAA+) proteins interacting with membranes.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • The fluorescent protein mEOS is a known reporter for ATPases Associated with diverse cellular Activities (AAA+) unfoldase activity.
  • Many AAA+ proteins are involved in processing membrane proteins, but studying them with mEOS has been challenging.
  • Directly fusing mEOS to membrane proteins abolishes its fluorescence, limiting its application.

Purpose of the Study:

  • To adapt the mEOS reporter system for studying AAA+ proteins that interact with membrane proteins.
  • To overcome the fluorescence quenching issue encountered when genetically fusing mEOS to membrane proteins.

Main Methods:

  • Purification of mEOS and AAA+ degrons, including a transmembrane domain.
  • Covalent linkage of mEOS and degrons using Sortase enzyme.
  • Assessment of mEOS fluorescence and photoconversion after linkage to membrane-associated domains.

Main Results:

  • The Sortase-mediated covalent linkage strategy successfully preserved mEOS fluorescence and photoconversion.
  • This method is effective even when mEOS is linked to a transmembrane domain.
  • The developed approach circumvents the fluorescence loss seen with direct genetic fusion.

Conclusions:

  • A broadly applicable platform has been established for studying membrane-associated AAA+ proteins.
  • This method enhances the utility of mEOS as a reporter for AAA+ unfoldase activity in membrane protein studies.
  • The innovative approach provides new possibilities for investigating the function of membrane-bound AAA+ proteins.
Keywords:
AAA+ proteinFluorescent proteinMembrane proteinProtein engineeringProteostasis

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