FIGURE 1.

Characterization of 601-177-12 nucleosomal arrays and recombinant HP1^Hsα. A, distribution of nucleosomes per array for histone H3K9Me3-containing and unmethylated arrays at 8 mm NaCl. Numbers of nucleosomes/per array were made from electron micrographs similar to those shown in Fig. 4, A and B. B, integrated sedimentation coefficient distribution of arrays possessing histone H3K9Me3 and unmethylated H3. Midpoint S values for the methylated and unmethylated arrays are 24.8S and 26.0S, respectively. Sedimentation values were calculated using the enhanced van Holde-Weischet method and normalized to standard conditions of pure water at 20 °C. C, HP1^Hsα binding to a histone H3K9me3 peptide was determined by fluorescence anisotropy for wild type, CD mutant, and CSD mutant proteins. K_D values for the wild type, CSD mutant, and CD mutant were 9 ± 2 μm, 24 ± 1 μm, and >500 μm, respectively. S.D. for each data point is shown. D, characterization of HP1^Hsα dimerization by equilibrium analytical ultracentrifugation. Wild type and mutant forms of HP1^Hsα were analyzed at nine different concentrations in triplicate. Shown are the fits and residuals for wild type HP1^Hsα, where the fits are forced to conform to either a dimeric species (top panels) or a monomeric species (bottom panels). Fits to the data are shown on the left; fit residuals are shown on the right. The expected molecular weight for the HP1^Hsα monomers is 25.4 kDa, and the best-fit molecular weight values of wild type, CD mutant, and CSD mutant HP1^Hsα proteins were 48.6, 45.2, and 22.6 KDa, respectively.