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, PPα, comprising a polyproline II helix, a loop, and an α helix. The redesign provides a de novo framework for interrogating noncovalent interactions. Optimized PPα has significantly improved thermal stability with a midpoint unfolding temperature (TM) of 51 °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-π 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.

Miniprotein Design: Past, Present, and Prospects.

The design and study of miniproteins, that is, polypeptide chains <40 amino acids in length that adopt defined and stable 3D structures, is resurgent. Miniproteins offer possibilities for reducing the complexity of larger proteins and so present new routes to studying sequence-to-structure and sequence-to-stability relationships in proteins generally. They also provide modules for protein design by pieces and, with this, prospects for building more-complex or even entirely new protein structures. In addition, miniproteins are useful scaffolds for templating functional domains, for example, those involved in protein-protein interactions, catalysis, and biomolecular binding, leading to potential applications in biotechnology and medicine. Here we select examples from almost four decades of miniprotein design, development, and dissection. Simply because of the word limit for this Account, we focus on miniproteins that are cooperatively folded monomers in solution and not stabilized by cross-linking or metal binding. In these cases, the optimization of noncovalent interactions is even more critical for the maintenance of the folded states than in larger proteins. Our chronology and catalogue highlights themes in miniproteins, which we explore further and begin to put on a firmer footing through an analysis of the miniprotein structures that have been deposited in the Protein Data Bank (PDB) thus far. Specifically, and compared with larger proteins, miniproteins generally have a lower proportion of residues in regular secondary structure elements (α helices, ß strands, and polyproline-II helices) and, concomitantly, more residues in well-structured loops. This allows distortions of the backbone enabling mini-hydrophobic cores to be made. This also contrasts with larger proteins, which can achieve hydrophobic cores through tertiary contacts between distant regions of sequence. On average, miniproteins have a higher proportion of aromatic residues than larger proteins, and specifically electron-rich Trp and Tyr, which are often found in combination with Pro and Arg to render networks of CH-π or cation-π interactions. Miniproteins also have a higher proportion of the long-chain charged amino acids (Arg, Glu, and Lys), which presumably reflects salt-bridge formation and their greater surface area-to-volume ratio. Together, these amino-acid preferences appear to support greater densities of noncovalent interactions in miniproteins compared with larger proteins. We anticipate that with recent developments such as parametric protein design, it will become increasingly routine to use computation to generate and evaluate models for miniproteins in silico ahead of experimental studies. This could include accessing new structures comprising secondary structure elements linked in previously unseen configurations. The improved understanding of the noncovalent interactions that stabilize the folded states of such miniproteins that we are witnessing through both in-depth bioinformatics analyses and experimental testing will feed these computational protein designs. With this in mind, we can expect a new and exciting era for miniprotein design, study, and application.

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 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 (TM) 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 TM 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.

Sterically geared tris-thioureas; transmembrane chloride transporters with unusual activity and accessibility.

Tris-N-arylthioureas derived in one step from 1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene are remarkably effective anion carriers. With optimised aryl substituents their activities come close to the best currently known, suggesting that they might find use as readily available standards in anion transport research.