Circular dichroism studies of DsrA and the DsrA:Hfq complexes. (A) Circular dichroism spectra of 0.5 μM DsrA with (▪) and without (♦) 3.0 μM Hfq, in 50 mM cacodylate buffer (pH 6.6), 250 mM NaCl at 37°C. The difference spectrum (▴) corresponds very closely to the spectrum of Hfq alone (data not shown). (B) Trace of circular dichroism intensity at 263 nm as a function of temperature (0°–100°C) for 0.5 μM DsrA and with (□) and without (○) 3.0 μM Hfq in 3 M urea, 50 mM cacodylate buffer (pH 6.6), and 250 mM NaCl. Lines represent fits of the data to a unimolecular two-state folding model. (C) Binding of DsrA to Hfq in the presence of urea and at high temperature. DsrA was incubated with or without 3.0 μM Hfq in 50 mM cacodylate buffer (pH 6.6), 250 mM NaCl, with 0, 3 M, or 7 M urea. Samples were incubated at 37°C or 65°C and run on gels at the same temperature. We observed 100% binding of DsrA to Hfq irrespective of the urea concentration or temperature of incubation.
