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

Isabella R Walter; Baylee A Smith; Dominic Castanzo; Matthew L Wohlever

doi:10.1016/j.pep.2025.106724

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

Protein Expr Purif. 2025 Aug:232:106724. doi: 10.1016/j.pep.2025.106724. Epub 2025 Apr 28.

Authors

Isabella R Walter¹, Baylee A Smith¹, Dominic Castanzo², Matthew L Wohlever³

Affiliations

¹ Department of Chemistry & Biochemistry, University of Toledo, Toledo, OH, 43606, USA.
² Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA; Institute of Quantitative Biosciences, University of California, Berkeley, CA, 94720, USA.
³ Department of Chemistry & Biochemistry, University of Toledo, Toledo, OH, 43606, USA; Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA, 15261, USA. Electronic address: wohlever@pitt.edu.

PMID: 40306474
DOI: 10.1016/j.pep.2025.106724

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 multiple different AAA+ degrons, including a transmembrane domain. We then covalently linked mEOS and the degrons via Sortase. This innovative approach preserves mEOS fluorescence and photoconversion, even upon linkage to a transmembrane domain. Together, this work offers a broadly applicable platform for the study of membrane associated AAA+ proteins.

Keywords: AAA+ protein; Fluorescent protein; Membrane protein; Protein engineering; Proteostasis.

MeSH terms

ATPases Associated with Diverse Cellular Activities* / chemistry
ATPases Associated with Diverse Cellular Activities* / genetics
ATPases Associated with Diverse Cellular Activities* / metabolism
Aminoacyltransferases / chemistry
Aminoacyltransferases / genetics
Aminoacyltransferases / metabolism
Bacterial Proteins* / chemistry
Bacterial Proteins* / genetics
Bacterial Proteins* / metabolism
Cysteine Endopeptidases / chemistry
Cysteine Endopeptidases / genetics
Cysteine Endopeptidases / metabolism
Escherichia coli / genetics
Escherichia coli / metabolism
Fluorescence
Luminescent Proteins* / chemistry
Luminescent Proteins* / genetics
Luminescent Proteins* / metabolism
Membrane Proteins* / chemistry
Membrane Proteins* / genetics
Membrane Proteins* / metabolism
Recombinant Fusion Proteins / chemistry
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism

Substances

ATPases Associated with Diverse Cellular Activities
Luminescent Proteins
Recombinant Fusion Proteins
Membrane Proteins
Bacterial Proteins
Aminoacyltransferases
Cysteine Endopeptidases