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. 2013 Oct 22;8(10):e76726. doi: 10.1371/journal.pone.0076726

Figure 7. Actin affinity and stability of AS-Cdc8p and mutants.

Figure 7

Actin affinity of AS-Cdc8p, wildtype and mutants, measured by cosedimentation as described in Methods (20 mM MOPS, pH 7.0, 150 mM NaCl, 2 mM MgCl2, 5 µM actin). AS-Wildtype, Kapp = 6.3×106 M−1 (n = 4); AS-16.30, Kapp = 2.6×106 M−1 (n = 3); the affinity AS-114.117.118 (n = 1) and AS-121.131.138 (n = 1) were too weak to measure. Inset: Actin affinity of AS-Cdc8p (filled circles) and unacetylated Cdc8p (open circles). The affinity of wildtype AS-Cdc8p is ∼500-fold greater than unacetylated Cdc8p. The affinities are too great and too weak, respectively, to calculate the Kapp. Binding conditions: Same as above except 20 mM MOPS, pH 7.0, 100 mM NaCl, 5 mM MgCl2 where Tm binds actin with higher affinity. B. Thermal stability determined by measuring the ellipticity at 222 nm between 0–60°C. The ellipticity was normalized to a scale of 0 to 1. The melting temperature (TM) is defined as the temperature where the normalized ellipticity is 0.5. The observed TM (n = 1) are: AS-WT = 33.1°C, AS-16.30 = 34.1°C; AS-114.117.118 = 31.5°C, AS-121.131.138 = 34.4°C.