Structural requirements and thermodynamics of the interaction of proline peptides with profilin

The binding to poly(l-proline) is used for the affinity purification of profilins, but little is known about the structural and thermodynamic aspects of the interaction. We used changes in the intrinsic fluorescence of profilin, CD spectroscopy, and isothermal titration calorimetry to assess how the size and composition of synthetic proline-rich peptides 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 ≥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 μM proline decamer units for amoeba profilin and 20−30 μM for human profilin. Consistent with a significant hydrophobic component of the interaction, ΔCp is negative and higher salt concentrations enhance the affinity. No protons dissociate or bind during the interaction. Binding of poly(l-proline) is favored both entropically and enthalpically. Substitution of glycine in proline undecamers reduces affinity by about 1 kcal mol-1 for each substitution due to increased rotational freedom of the free peptides. Substitution of alanine has a similar effect. Disorder in the free peptides imparts an unfavorable entropic cost for immobilizing the substituted peptides on the binding site on profilin