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Figure 4. CAF-1 binds (H3-H4)2 tetramers and H3-H4 dimers with similar affinities, yet 2-fold different stoichiometries. (A) An overlay of the binding curves for CAF-1 titrated into a H3(L126R, I130R)-H4 double mutant that effectively prevents tetramer formation (gray squares) and a cross-linked (H3-H4)2 tetramer (triangle) with the wild-type H3-H4 curve from Figure 1 (dashed line). The curves are very similar to wild-type and produce dissociation constants of 5.5 nM for the double mutant and 9.0 nM for the cross-linked complex (Table 1). (B) Determination of the stoichiometry of the CAF-1•WT H3-H4 complex by titration of CAF-1 into a constant concentration of labeled H3-H4. Fluorescence change occurs until wild-type H3-H4 is saturated with CAF-1 and the ratio at this inflection point is equal to the number of CAF-1 molecules bound to a single H3-H4 complex. (C) Determination of the stoichiometry of CAF-1/H3(L126R, I130R)-H4 complex. The fluorescence change plateaus similar to that of wild-type (H3-H4)2 tetramer and corresponds to 0.5 CAF-1 molecules per 1 H3(L126R, I130R)-H4 dimer or a 1:2 stoichiometry. (D) The cross-linked (H3-H4)2 tetramer is bound by a single CAF-1 molecule, as shown by the near 1:1 ratio obtained by the titration.The error bars represent the standard error within individual data points.The total data points for a single experiment are 48. Image published in: Winkler DD et al. (2012) © The Author(s) 2012. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license Larger Image Printer Friendly View

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