Engineering protein stability with atomic precision in a monomeric miniprotein

Abstract

Miniproteins simplify the protein-folding problem, allowing the dissection of forces that stabilize protein structures. Here we describe PPα-Tyr, a designed peptide comprising an α-helix buttressed by a polyproline II helix. PPα-Tyr is water soluble and monomeric, and it unfolds cooperatively with a midpoint unfolding temperature (T) of 39 °C. NMR structures of PPα-Tyr reveal proline residues docked between tyrosine side chains, as designed. The stability of PPα 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 to 20 °C. We attribute this result to the loss of CH-π 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-π interactions in biologically important complexes between proline-rich ligands and SH3 and similar domains.

A Comprehensive Analysis of Anion–Quadrupole Interactions in Protein Structures

“…Highly conserved anion–quadrupole interactions can be observed, even among remote homologs, suggesting that such interactions could contribute to folding and stability of proteins. The recent success in designing miniature monomeric proteins based on conserved and stabilizing structural motifs (e.g., cation–π in TrpPlexus 81 , CH–π in PPα-Tyr 29 ) suggests that the conserved anion–quadrupole structural motifs observed here could also be exploited for similar design studies. In general, standard protein design algorithms typically lack energy functions for interactions such as cation–π or water-mediated stabilization.…”

Hyperthermostable cube-shaped assembly in water

“…2 is placed between the two positively charged N-methylpyridinium rings to generate cation-π interactions ( Fig. 1c, d), which are often seen in binding sites relating to positively charged species in biological systems [33][34][35] .…”

Stabilizing and Understanding a Miniprotein by Rational Redesign

“…In the latter, protons of C-H bonds interact with aromatic rings. [17][18][19][20] Here, we explore the optimization of PP by rational redesign, revealing sequence-to-stability relationships for the miniprotein fold and delivering a de novo framework of significantly enhanced thermal stability.…”