Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase

• Published in: Scientific reports Vol. 7; no. 1; p. 40948
• Main Authors: Ibrahim, Ziad, Martel, Anne, Moulin, Martine, Kim, Henry S., Härtlein, Michael, Franzetti, Bruno, Gabel, Frank
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 19.01.2017; Nature Publishing Group
• Subjects: 631/535/1261; 631/57/2272/2273; Archaea - enzymology; ATPases Associated with Diverse Cellular Activities - chemistry; ATPases Associated with Diverse Cellular Activities - metabolism; Biochemistry, Molecular Biology; Deuterium; Fluorescence spectroscopy; Green fluorescent protein; Green Fluorescent Proteins - metabolism; Humanities and Social Sciences; Labeling; Life Sciences; Macromolecules; multidisciplinary; Neutron scattering; Neutrons; Protein Conformation; Protein Folding; Proteins; Scattering, Small Angle; Science; Stroke; Structural Biology; Substrates; Temperature effects
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep40948

We present a combination of small-angle neutron scattering, deuterium labelling and contrast variation, temperature activation and fluorescence spectroscopy as a novel approach to obtain time-resolved, structural data individually from macromolecular complexes and their substrates during active biochemical reactions. The approach allowed us to monitor the mechanical unfolding of a green fluorescent protein model substrate by the archaeal AAA+ PAN unfoldase on the sub-minute time scale. Concomitant with the unfolding of its substrate, the PAN complex underwent an energy-dependent transition from a relaxed to a contracted conformation, followed by a slower expansion to its initial state at the end of the reaction. The results support a model in which AAA ATPases unfold their substrates in a reversible power stroke mechanism involving several subunits and demonstrate the general utility of this time-resolved approach for studying the structural molecular kinetics of multiple protein remodelling complexes and their substrates on the sub-minute time scale.