Analysis of the chaperonin cycle using CP86 variants.

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.

A. 对GroEL CP86-RW在添加1 mM ATP后的停止流动分析。黑色轨迹对应于GroEL R231W，表示野生型类似行为。灰色轨迹对应于GroEL CP86-RW。在本图及所有后续涉及将原始数据拟合至理论曲线的图中，下方面板总结了每个拟合产生的残差，图例与上方面板对应。B. 在添加ATP和等摩尔GroES后对GroEL CP86-RW的停止流动分析。图例与A相同。C. 对氧化和还原状态下的GroEL CP86-C4结合的罗丹明分子进行蛋白酶K保护实验。所示样品为在25°C下用1 µg/ml蛋白酶K消化30分钟后，浓缩于Amicon Ultra-0.5 100K离心滤器单元的溶液。箭头表示罗丹明的位置。凝胶底部星号指示随后用于制备在D面板中分析的负染色样品的样品。D. 使用还原状态的GroEL CP86-C4制备的BeFx稳定化、蛋白酶K处理的GroE-罗丹明三元复合物的电子显微镜图像。标尺条表示100纳米。