Fig 2. DNA binding properties of Pf-SSB^MDCC.

(A) Purified Pf-SSB^MDCC was analyzed on a 12% SDS-PAGE gel and imaged after staining with coommassie dye or detected using fluorescence imaging. M denotes the protein ladder. (B) Excitation (blue, λ_ex) and emission (green, λ_em) spectra of 1 μM Pf-SSB^MDCC are shown. The dotted lines correspond to an excitation and emission maxima of 430 nm and 482 nm, respectively. (C) 1 μM Pf-SSB^MDCC was excited at 430 nm and emission spectra were measured in the absence of DNA (grey) and in the presence of a 125 bp dsDNA (blue) or an oligo-dT 70 nt ssDNA (red). A four-fold increase in Pf-SSB^MDCC fluorescence is observed in the presence of the (dT)₇₀ ssDNA oligonucleotide. (D) Fluorescence titration of Pf-SSB^MDCC with increasing concentrations of ssDNA [(dT)₇₀]. Pf-SSB^MDCC binds stoichiometrically, with one SSB tetramer binding to one (dT)₇₀ oligonucleotide as denoted by the dotted line. The mean values and standard errors from three independent experiments are shown. (E) Isothermal calorimetric measurement of changes in enthalpy associated with binding of two (dT)₃₅ molecules to Pf-SSB and Pf-SSB^MDCC are shown. Both proteins bind stoichiometrically with similar observed heat changes ΔH_obs = -73.1±0.2 kcal mol^-1 and -71.8±0.2 kcal mol^-1 for Pf-SSB and Pf-SSB^MDCC, respectively. The mean values and standard errors from three independent experiments are shown.