Figure 1. Preparation and biochemical characterization of CP2c(63-302). (A) Schematic diagram showing the putative domain organization of CP2c.2 Constructed region in this study and the known binding site of the dodecapeptide are illustrated by arrows. TAD, transactivation domain; BD, basic domain; TD, tetramerization domain. (B) Gel-filtration elution profile of the purified CP2c(63-302). Inset shows SDS-PAGE image for the elution fractions containing the protein. (C) Hydrodynamic size of CP2c(63-302) as estimated by analytical gel-filtration. Black dots indicate the protein standards used. Red dot designates the Kav value of CP2c(63-302), of which apparent molecular weight was calculated using the standard curve.9,10
Figure 2. Examination of a dodecapeptide binding to CP2c(63-302). (A) Thermal denaturation of CP2c(63-302) (20 μM), monitored by CD at 222 nm, in the absence (black) and presence of the binding peptide (blue and red for 1 and 2 equivalent titration, respectively). Inset shows standard far-UV CD spectra in the absence (black) and presence (green) of 2 equivalents of the peptide. (B) 2D-[1H/15N]-TROSY NMR spectra of [15N]CP2c(63-302) (250 μM), in the absence (black) and presence of 2 equivalents of the binding peptide (green).
Figure 3. Examination of zinc binding to CP2c(63-302). (A) 2D-[1H/15N]-TROSY NMR spectra of [15N]CP2c(63-302) (250 μM), in the absence (black) and presence of 2 equivalents of zinc (red). Inset shows standard far-UV CD spectra in the absence (black) and presence (red) of 2 equivalents of zinc. (B) Thermal denaturation of CP2c(63-302) (20 μM), monitored by CD at 222 nm, in the absence (black) and presence of zinc (molar ratios are indicated for individual colors).
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