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Analysis of the chaperonin cycle using CP86 variants.

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posted on 30.10.2013, 19:52 authored by Toshifumi Mizuta, Kasumi Ando, Tatsuya Uemura, Yasushi Kawata, Tomohiro Mizobata

A. Stopped-flow analysis of GroEL CP86-RW upon addition of 1 mM ATP. Black traces correspond to GroEL R231W and denote wild-type like behavior. Gray traces correspond to GroEL CP86-RW. In this and all subsequent figures that involve fits of raw data to theoretical curves, the lower panel summarizes the residuals derived from each fit, with legends corresponding to the upper panel. B. Stopped-flow analysis of GroEL CP86-RW upon addition of ATP and equimolar GroES. Legends are as in A. C. Proteinase K protection assays of rhodanese molecule bound to oxidized and reduced GroEL CP86-C4. Samples shown are from solutions concentrated on an Amicon Ultra-0.5 100K centrifugal filter unit after a 30 min digestion with 1 µg/ml Proteinase K at 25°C. The arrowhead denotes the position of rhodanese. The asterisk at the bottom of the gel indicates the sample that was subsequently used to prepare negatively stained samples analyzed in panel D. D. Electron micrographs of BeFx-stabilized, Proteinase K-treated samples of GroE-rhodanese ternary complexes prepared using reduced GroEL CP86-C4. Scale bar denotes 100 nm.