Figure 3

MmCpn•Cys-0 can effectively mediate substrate folding. (A) Scheme of chaperonin folding cycle (B) Non-denaturing agarose electrophoresis to monitor binding MmCpn and the substrate protein rhodanese. Cy3-maleimide labeled rhodanese co-migration with MmCpn in different nucleotide states (top). Total protein stained with Sypro Ruby. (C) Protease protection of Cy3-rhodanese by MmCpn in different nucleotide states.Rho: Rhodanese; *: background band. (D) Rhodanese folding of WT and Cys-0 MmCpn. The y-axis represents the amount of cyanide converted to thiocyanate by rhodanese in 10 minutes relative to the mean amount converted by the wild type sample with ATP-AlFx. Cys-0 and WT do not significantly differ under actively cycling conditions (p-value by two sided t-test for ATP: p = 0.134) but Cys-0 folds rhodanese with slightly higher yield than wild type chaperonin under ATP-AlFx conditions (p-value for ATP*AlFx: p = 0.0409). This is based on n = 3 measurements for both conditions.