Stabilizing and Understanding a Miniprotein by Rational Redesign

Miniproteins reduce the complexity of the protein-folding problem allowing systematic studies of contributions to protein folding and stabilization. Here, we describe the rational redesign of a miniprotein, PP alpha, comprising a polyproline II helix, a loop, and an alpha helix. The redesign provides a de novo framework for interrogating noncovalent interactions. Optimized PP alpha has significantly improved thermal stability with a midpoint unfolding temperature (T-M) of 51 degrees C. Its nuclear magnetic resonance structure indicates a density of stabilizing noncovalent interactions that is higher than that of the parent peptide, specifically an increased number of CH-pi interactions. In part, we attribute this to improved long-range electrostatic interactions between the two helical elements. We probe further sequence-stability relationships in the miniprotein through a series of rational mutations.