TY - JOUR

T1 - A real-time analysis of GFP unfolding by the AAA+ unfoldase PAN

AU - Krüger, Georg

AU - Kirkpatrick, John

AU - Mahieu, Emilie

AU - Franzetti, Bruno

AU - Gabel, Frank

AU - Carlomagno, Teresa

N1 - Funding Information: This work has been supported by the DFG (grant CA294/13-1 to TC) and a Leverhulme International Professorship to TC. The authors thank Susanne zur Lage at HZI, Braunschweig for support with sample preparation.

PY - 2023/5

Y1 - 2023/5

N2 - 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.

AB - 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.

KW - AAA+ ATPase

KW - GFP

KW - PAN

KW - Protein unfolding

KW - Real-time NMR spectroscopy

UR - http://www.scopus.com/inward/record.url?scp=85152225287&partnerID=8YFLogxK

U2 - 10.1016/j.jmr.2023.107431

DO - 10.1016/j.jmr.2023.107431

M3 - Article

C2 - 37058954

AN - SCOPUS:85152225287

VL - 350

JO - Journal of magnetic resonance

JF - Journal of magnetic resonance

SN - 1090-7807

M1 - 107431

ER -