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Thermal-induced unfolding measurements followed by DSC.

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posted on 19.02.2013, 19:15 authored by Júlio C. Borges, Thiago V. Seraphim, David Z. Mokry, Fabio C. L. Almeida, Douglas M. Cyr, Carlos H. I. Ramos

(A) Sis1; (B) Sis1_Δ124–174; (C) Sis1_Δ121–257 and (D) SYS. The figures represent the first and second scans after baseline treatment. Sis1 and Sis1_Δ124–174 showed two overlapping transitions with similar Tms of approximately 59 and 67°C (Table 1). Sis1_Δ121–257 and SYS showed two well separated unfolding transitions where the second Tm was within 64–66°C (Table 1). For all proteins, the second unfolding transition was more than 95% reversible. The first unfolding transition of Sis1, Sis1_Δ124–174 and Sis1_Δ121–257 was approximately 90% reversible when the proteins were heated to 90°C. SYS (Fig. D) presented a reversibility of approximately 60% when heated to 90°C. However, when SYS was heated to 50°C (Fig. D, inset) it was more than 95% reversible. Similar behaviors were observed with other proteins. E–F) Kirchoff plots showing the dependence of ΔHcal with Tm for Sis1 and its mutants. Urea concentrations from 0.25 M to 2.0 M were used in order to disturb both ΔHCal and Tm that were monitored by DSC. The figure shows the Kirchoff plot for the first (E) and second (F) thermal-induced unfolding transition. The curves were fitted using Eq. 3 in order to obtain the ΔCp for each unfolding transition.