Direct Calorimetric Determination of a Complete Polyproline II (pII) Propensity Scale Reveals PII Enhancement in Intrinsically Disordered Proteins

Abstract

Circular dichroism spectropolarimetry experiments and other studies have suggested that the denatured states of polypeptides may comprise a smaller conformational space than previously thought, and that the polyproline II (PII) helix is one of the conformations that is highly populated in the denatured state. Src-homology 3 (SH3) is a modular domain that recognizes and binds peptide in the PII conformation, such as SosY, with high specificity. Our experimental strategy is to use isothermal titration calorimetry (ITC) to monitor the binding of SH3 to engineered SosY peptides, enabling the direct detection of ligand population that is in the PII conformation. Importantly, our model system allows direct access to free energy information concerning the context dependent PII propensity of amino acids at specific sites in the SosY peptide. Binding isotherms have been measured and quantitative estimates made for the PII propensity all twenty amino acids, developing a complete PII scale. Correlations of the calorimetrically determined scale to the GenomeNet database of amino acid physico-chemical property and secondary structure propensity scales revealed no significant correlations. An algorithm is developed to calculate the average PII propensities of different sequences, revealing that intrinsically disordered proteins have an enhancement of PII compared to sequences that fold. Upon random shuffling, the high PII regions within intrinsically disordered segments are lost, demonstrating local enrichment of high PII bias within these sequences.