Figure 3. Zinc slows the dissociation rate of the high-affinity RsrA^red.Zn²⁺-σ^R complex.

Conditions used for all experiments were 50 mM Tris (pH 7.5) buffer containing 100 mM NaCl and 2 mM DTT. Temperature was either 35 °C (a,b) or 25 °C (c,d). (a) Competition ITC data for RsrA^red.Zn²⁺ (100 μM) binding σ^R (10 μM, cell concentration) in the presence of RsrA* Cys11Ser Cys44Ser (50 μM). RsrA* Cys11Ser Cys44Ser binds σ^R with a weaker affinity than wild-type RsrA (Supplementary Table 1). Control data show titration of RsrA^red.Zn²⁺ into buffer containing 50 μM RsrA* Cys11Ser Cys44Ser. Fitted parameters for a competitive binding site model from three independent measurements were N=1.01±0.05, K_d=0.78±0.034 nM, ΔH=−23.05±1.10 kcal mol⁻¹, ΔS=−38.70±3.10 cal mol⁻¹ deg⁻¹. (b) Direct ITC data for RsrA^red (100 μM) binding σ^R (10 μM) in the absence of zinc. Control data show titration of RsrA^red into buffer. Fitted parameters for a single site-binding model were N=0.97±0.004, K_d=79.3±4.2 nM, ΔH=−16.26±0.13 kcal mol⁻¹, ΔS=−20.3±4.34 cal mol⁻¹ deg⁻¹. (c) Main panel: tryptophan emission fluorescence stopped-flow association data for RsrA^red.Zn²⁺ binding σ^R under pseudo-first-order conditions (λ_ex, 295 nm). Residuals to the fit of a single exponential are shown below the panel. Inset: pseudo-first-order plot of observed rates (k_obs) as a function of RsrA concentration in the presence and absence of stoichiometric zinc (with associated error bars). See Supplementary Table 2 for derived values of k_on. Error bars are within the data point symbols. (d) Dissociation of the RsrA^red–σ^R complex measured by competition stopped-flow in which a 10-fold excess of σ^R Trp88Ile Trp119Ile was used to displace wild-type σ^R (Methods). Main panel shows data for the RsrA^red–σ^R complex in the absence of bound zinc. Inset: dissociation data for the RsrA^red–σ^R complex in the presence of 1 and 3 equiv. of zinc (open and filled triangles, respectively). See Supplementary Table 2 for values of k_off.