Massachusetts Institute of Technology
Thursday, Apr. 5, 2012
Refreshments: 3:30 pm in 219 JFB
Lecture 4:00pm (102 JFB)
Title: Single-molecule Studies of ClpXP Machinery Fully Loaded
ClpX is a ring-shaped molecular motor involved in cellular protein degradation. Fueled by ATP, ClpX works in tandem with protease ClpP to recognize, unfold, translocate and finally cleave protein substrates. ClpX is part of the versatile AAA+ family, which carry common structural features while supporting diverse cellular processes. ClpXP activity is probed at the single molecule level using both a single molecule fluorescence assay and a loaded assay employing an optical double-trap geometry for low noise, passive-force clamping. In the single molecule fluorescence assay, ClpXP activity is monitored using a series of fluorescently labeled substrates. In the fully loaded assay, a single-chain ClpXP motor with a poly-linked substrate engaged into its pore is tethered between two trapped polystyrene beads held in a dumbbell configuration. The variation in substrate length is monitored throughout degradation by tracking bead separation allowing for direct measurement of ClpXP machinery including: unfolding times, translocation velocity and translocation stepping.