STRUCTURAL REQUIREMENTS AND THERMODYNAMICS OF THE INTERACTION OF PROLINE PEPTIDES WITH PROFILIN

The binding to poly(L-proline) is used for the affinity purification o f profilins, but little is known about the structural and thermodynami c aspects of the interaction. We used changes in the intrinsic fluores cence of profilin, CD spectroscopy, and isothermal titration calorimet ry to assess how the size and composition of synthetic proline-rich pe ptides influence binding to Acanthamoeba and human profilins. Although a 6 residue type II poly(L-proline) helix can span the binding site, highest affinity binding is achieved by proline oligomers greater than or equal to 10 residues. Binding is stereospecific since (D-proline)( 11) does not bind. In 75 mM KCl the dissociation equilibrium constant for poly(L-proline) is about 10 mu M proline decamer units for amoeba profilin and 20-30 mu M for human profilin. Consistent with a signific ant hydrophobic component of the interaction, Delta C-p is negative an d higher salt concentrations enhance the affinity. No protons dissocia te or bind during the interaction. Binding of poly(L-proline) is favor ed both entropically and enthalpically. Substitution of glycine in pro line undecamers reduces affinity by about 1 kcal mol(-1) for each subs titution due to increased rotational freedom of the free peptides. Sub stitution of alanine has a similar effect. Disorder in the free peptid es imparts an unfavorable entropic cost for immobilizing the substitut ed peptides on the binding site on profilin.