Engineering protein stability with atomic precision in a monomeric miniprotein

Miniproteins simplify the protein-folding problem, allowing the dissection of forces that stabilize protein structures. Here we describe PP alpha-Tyr, a designed peptide comprising an alpha-helix buttressed by a polyproline II helix. PP alpha-Tyr is water soluble and monomeric, and it unfolds cooperatively with a midpoint unfolding temperature (T-M) of 39 degrees C. NMR structures of PP alpha-Tyr reveal proline residues docked between tyrosine side chains, as designed. The stability of PP alpha is sensitive to modifications in the aromatic residues: replacing tyrosine with phenylalanine, i.e., changing three solvent-exposed hydroxyl groups to protons, reduces the T-M to 20 degrees C. We attribute this result to the loss of CH-pi interactions between the aromatic and proline rings, which we probe by substituting the aromatic residues with nonproteinogenic side chains. In analyses of natural protein structures, we find a preference for proline-tyrosine interactions over other proline-containing pairs, and observe abundant CH-p interactions in biologically important complexes between proline-rich ligands and SH3 and similar domains.