Role of denatured state properties in chaperonin action probed by single- molecule spectroscopy

Preparation and labeling of proteins. The preparation of GroEL-SR1, GroES, fluorescently labeled rhodanese and cyclophilin A has been described previously (1, 2). Table S1 summarizes the sequences of the variants used in this study. For carbamidomethylation of donorand acceptor labeled Rho, we transferred the corresponding Rho-variant into 8M Urea, 0.6 M TrisHCl, 5mM EDTA, 0.01% Tween 20 pH8 (CAM-buffer 1) using a High-Trap desalting column (GE Healthcare). The protein-containing fractions were pooled and subsequently reduced by the addition of 2-mercaptoethanol (100 mM in 500 μl of protein solution). Carbamidomethylation was performed by the addition of 50 μl of CAM-reagent (300 mg iodoacetamide in 500 μl 1 M TrisHCl, 5 mM EDTA, pH8.5 heated to 60°C; after cooling, 50 μl of 0.1 M ascorbic acid in CAM-buffer 1 were added). After 5 minutes at room temperature, the mixture was applied to a desalting column (GE Healthcare) pre-equilibrated with 6 M GdmCl, 0.1 M KH2PO4, 1mM EDTA pH 7). The pooled fractions were stored at 80°C.