Figure 7: Influence of complex formation on the kinetics of cluster formation and on the complex affinities.
(a) Kinetics rates of Fe–S cluster formation on IscU. The experiments are all conducted under the same conditions (1 μM IscS or IscS mutants, 25 μM Fe2+, 50 μM IscU, 250 μM Cys, 2 mM DTT and 5 μM of CyaY or CyaY mutants). Green curve: control experiment in the absence of CyaY; red curve: control experiment in the presence of wild-type CyaY; blue, magenta, orange and cyan curves: experiments carried out by substituting wild-type proteins with CyaY_E18K_E19K_E22K, IscS_R220E_R223E_R225E, CyaY_W61R and IscS_I314E_M315E, respectively. (b) Biolayer interferometry profile of the titration of CyaY with increasing concentrations of IscS in the absence of IscU. (c) As in b, but in the presence of IscU. Because the complex is stabilized, lower concentrations of IscS are needed to reach saturation. (d) Plot of the interferometry response obtained by immobilizing IscS as a function of increasing concentrations of cluster-free (circle) and cluster-loaded (diamond) IscU_D39A mutant.
