Figure 3.

Characterization of RTX polymers. The Ca²⁺-dependent aggregation of the RTX domain was evaluated using ThT fluorescence. (A) The purified RTX domain was refolded into buffers containing increasing concentrations of Ca²⁺, and emission spectra of ThT fluorescence were collected after overnight incubation. Increasing Ca²⁺ concentrations resulted in decreasing maximal emission intensities [0 (●), 20 (▽), 60 (■), and 200 μM Ca²⁺ (◇)]. Background fluorescence of buffer without RTX protein was subtracted from each of the emission spectra. (B) The kinetics of ThT fluorescence were monitored by emission at 482 nm as a function of time in the presence or absence of Ca²⁺ and Mg²⁺. RTX protein in the absence of Ca²⁺ showed an increase in ThT fluorescence (black circles), as did the protein incubated in Mg²⁺ (red circles). This increase in TfT fluorescence was not observed with the Ca²⁺-bound protein (yellow triangles) or in buffer controls (green triangles). (C) Analytical gel filtration was used to assess the soluble, monomeric RTX protein in solution through the time course of aggregation. The level of soluble RTX protein in the absence of Ca²⁺ decreased as a function of incubation time (○), as did the level of protein incubated in 2 mM Mg²⁺ (■). Calcium-bound (2 mM) RTX protein remained soluble and monomeric over the incubation times tested (●).