Fig. 5. Dimerization of the DBD of PU.1 is electrostatically mediated and conformationally destabilizing.

(A) CD-detected titration of ΔN167 at 50 mM NaCl from 25 to 800 μM. (B) Analysis of the titration by singular value decomposition yields a two-state transition with a dissociation constant K₂ of 202 ± 72 μM. (C) ¹H-¹⁵N HSQC as a function of salt from 25 to 500 mM NaCl. Residues with the strongest ¹H-¹⁵N CSPs (Y173, M223, G239, V244, and L248) are boxed. Inset: Salt dependence of the CSPs of these residues. (D) Summary of the residue CSPs with the average %SASA from the unbound NMR monomer, 5W3G. Residues above a 0.5-ppm cutoff are colored in dark blue, and the subset of internal residues (<35% SASA, based on the termini) is marked with yellow circles. Residues implicated in the DNA-bound dimer are marked with green circles. (E) Mapping of the high-CSP residues to 5W3G. Green residues mark known residues involved in the DNA-bound dimer (20). (F) Chemical shift–derived secondary structure prediction via the CSI using ¹H and ¹⁵N signals. The color scheme follows the HSQC in (C). Regions with significant changes in secondary structure are marked by arrows. (G) Near-UV CD-detected thermal melting of ΔN117 and ΔN165. Two salt concentrations were evaluated for ΔN165 (blue and gray). Inset: Representative near-UV CD spectra. (H) DSC thermograms (solid) for ΔN165 under conditions (salt and concentrations) in which the protein was primarily monomeric or dimeric. The ΔC_p values are given in kJ (mol monomer)⁻¹ K⁻¹. Dashed curves represent the two-state transition for a monomer (black) and dimer (blue). (I) Trp fluorescence-detected denaturation by urea of ΔN117 and ΔN165 at two monomer concentrations. Curves represent fit to the linear extrapolation model for a two-state dimer. The marked concentrations represent urea concentration at 50% unfolding.