title: Studying chaperone-proteases using a real-time approach based on FRET.
abstract: Chaperone-proteases are responsible for the processive breakdown of proteins in eukaryotic, archaeal and bacterial cells. They are composed of a cylinder-shaped protease lined on the interior with proteolytic sites and of ATPase rings that bind to the apical sides of the protease to control substrate entry. We present a real-time FRET-based method for probing the reaction cycle of chaperone-proteases, which consists of substrate unfolding, translocation into the protease and degradation. Using this system we show that the two alternative bacterial ClpAP and ClpXP complexes share the same mechanism: after initial tag recognition, fast unfolding of substrate occurs coinciding with threading through the chaperone. Subsequent slow substrate translocation into the protease chamber leads to formation of a transient compact substrate intermediate presumably close to the chaperone-protease interface. Our data for ClpX and ClpA support the mechanical unfolding mode of action proposed for these chaperones. The general applicability of the designed FRET system is demonstrated here using in addition an archaeal PAN-proteasome complex as model for the more complex eukaryotic proteasome.
authors: ETH Zürich, Institute of Molecular Biology & Biophysics, Switzerland.
authors: Kristina
authors: K
authors: Kolygo
authors: Namit
authors: N
authors: Ranjan
authors: Wolfgang
authors: W
authors: Kress
authors: Frank
authors: F
authors: Striebel
authors: Kaspar
authors: K
authors: Hollenstein
authors: Kai
authors: K
authors: Neelsen
authors: Miriam
authors: M
authors: Steiner
authors: Heike
authors: H
authors: Summer
authors: Eilika
authors: E
authors: Weber-Ban
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D049071:Endopeptidase Clp / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008954:Models, Biological; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D049071:Endopeptidase Clp