A real-time analysis of GFP unfolding by the AAA+ unfoldase PAN

Georg Krüger, John P. Kirkpatrick, Emilie Mahieu, Bruno Franzetti, Frank Gabel, Teresa Carlomagno*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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Abstract

Protein quality control systems are essential to maintain a healthy proteome. They often consist of an unfoldase unit, typically an AAA+ ATPase, coupled with a protease unit. In all kingdoms of life, they function to eliminate misfolded proteins, and thus prevent that their aggregates do harm to the cell, and to rapidly regulate protein levels in the presence of environmental changes. Despite the huge progress made in the past two decades in understanding the mechanism of function of protein degradation systems, the fate of the substrate during the unfolding and proteolytic processes remains poorly understood. Here we exploit an NMR-based approach to monitor GFP processing by the archaeal PAN unfoldase and the PAN-20S degradation system in real time. We find that PAN-dependent unfolding of GFP does not involve the release of partially-folded GFP molecules resulting from futile unfolding attempts. In contrast, once stably engaged with PAN, GFP molecules are efficiently transferred to the proteolytic chamber of the 20S subunit, despite the only weak affinity of PAN for the 20S subunit in the absence of substrate. This is essential to guarantee that unfolded but not proteolyzed proteins are not released into solution, where they would form toxic aggregates. The results of our studies are in good agreement with previous results derived from real-time small-angle-neutron-scattering experiments and have the advantage of allowing the investigation of substrates and products at amino-acid resolution.

Original languageEnglish
Article number107431
Number of pages10
JournalJournal of Magnetic Resonance
Volume350
Early online date5 Apr 2023
DOIs
Publication statusPublished - May 2023

Bibliographical note

Copyright © 2023 The Author(s). Published by Elsevier Inc.

Keywords

  • AAA+ ATPase
  • Protein unfolding
  • Real-time NMR spectroscopy
  • PAN
  • GFP

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