Design and Selection of Heterodimerizing Helical Hairpins for Synthetic Biology.

Synthetic biology applications would benefit from protein modules of reduced complexity that function orthogonally to cellular components. As many subcellular processes depend on peptide-protein or protein-protein interactions, de novo designed polypeptides that can bring together other proteins controllably are particularly useful. Thanks to established sequence-to-structure relationships, helical bundles provide good starting points for such designs. Typically, however, such designs are tested in vitro and function in cells is not guaranteed. Here, we describe the design, characterization, and application of de novo helical hairpins that heterodimerize to form 4-helix bundles in cells. Starting from a rationally designed homodimer, we construct a library of helical hairpins and identify complementary pairs using bimolecular fluorescence complementation in E. coli. We characterize some of the pairs using biophysics and X-ray crystallography to confirm heterodimeric 4-helix bundles. Finally, we demonstrate the function of an exemplar pair in regulating transcription in both E. coli and mammalian cells.

From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles.

The design of completely synthetic proteins from first principles-de novo protein design-is challenging. This is because, despite recent advances in computational protein-structure prediction and design, we do not understand fully the sequence-to-structure relationships for protein folding, assembly, and stabilization. Antiparallel 4-helix bundles are amongst the most studied scaffolds for de novo protein design. We set out to re-examine this target, and to determine clear sequence-to-structure relationships, or design rules, for the structure. Our aim was to determine a common and robust sequence background for designing multiple de novo 4-helix bundles. In turn, this could be used in chemical and synthetic biology to direct protein-protein interactions and as scaffolds for functional protein design. Our approach starts by analyzing known antiparallel 4-helix coiled-coil structures to deduce design rules. In terms of the heptad repeat, abcdefg -i.e., the sequence signature of many helical bundles-the key features that we identify are: a = Leu, d = Ile, e = Ala, g = Gln, and the use of complementary charged residues at b and c. Next, we implement these rules in the rational design of synthetic peptides to form antiparallel homo- and heterotetramers. Finally, we use the sequence of the homotetramer to derive in one step a single-chain 4-helix-bundle protein for recombinant production in E. coli. All of the assembled designs are confirmed in aqueous solution using biophysical methods, and ultimately by determining high-resolution X-ray crystal structures. Our route from peptides to proteins provides an understanding of the role of each residue in each design.

Generalized Born Implicit Solvent Models Do Not Reproduce Secondary Structures of De Novo Designed Glu/Lys Peptides.

We test a range of standard generalized Born (GB) models and protein force fields for a set of five experimentally characterized, designed peptides comprising alternating blocks of glutamate and lysine, which have been shown to differ significantly in α-helical content. Sixty-five combinations of force fields and GB models are evaluated in >800 µs of molecular dynamics simulations. GB models generally do not reproduce the experimentally observed α-helical content, and none perform well for all five peptides. These results illustrate that these models are not usefully predictive in this context. These peptides provide a useful test set for simulation methods.

Scalable synthesis and coupling of quaternary α-arylated amino acids: α-aryl substituents are tolerated in α-helical peptides.

Quaternary amino acids are important tools for the modification and stabilisation of peptide secondary structures. Here we describe a practical and scalable synthesis applicable to quaternary alpha-arylated amino acids (Q4As), and the development of solid-phase synthesis conditions for their incorporation into peptides. Monomeric and dimeric α-helical peptides are synthesised with varying degrees of Q4A substitution and their structures examined using biophysical methods. Both enantiomers of the Q4As are tolerated in folded monomeric and oligomeric α-helical peptides, with the (R)-enantiomer slightly more so than the (S).

Peptide Assembly Directed and Quantified Using Megadalton DNA Nanostructures.

In nature, co-assembly of polypeptides, nucleic acids, and polysaccharides is used to create functional supramolecular structures. Here, we show that DNA nanostructures can be used to template interactions between peptides and to enable the quantification of multivalent interactions that would otherwise not be observable. Our functional building blocks are peptide-oligonucleotide conjugates comprising de novo designed dimeric coiled-coil peptides covalently linked to oligonucleotide tags. These conjugates are incorporated in megadalton DNA origami nanostructures and direct nanostructure association through peptide-peptide interactions. Free and bound nanostructures can be counted directly from electron micrographs, allowing estimation of the dissociation constants of the peptides linking them. Results for a single peptide-peptide interaction are consistent with the measured solution-phase free energy; DNA nanostructures displaying multiple peptides allow the effects of polyvalency to be probed. This use of DNA nanostructures as identifiers allows the binding strengths of homo- and heterodimeric peptide combinations to be measured in a single experiment and gives access to dissociation constants that are too low to be quantified by conventional techniques. The work also demonstrates that hybrid biomolecules can be programmed to achieve spatial organization of complex synthetic biomolecular assemblies.

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.

Dynamic ion pair behavior stabilizes single α-helices in proteins.

Ion pairs are key stabilizing interactions between oppositely charged amino acid side chains in proteins. They are often depicted as single conformer salt bridges (hydrogen-bonded ion pairs) in crystal structures, but it is unclear how dynamic they are in solution. Ion pairs are thought to be particularly important in stabilizing single α-helix (SAH) domains in solution. These highly stable domains are rich in charged residues (such as Arg, Lys, and Glu) with potential ion pairs across adjacent turns of the helix. They provide a good model system to investigate how ion pairs can contribute to protein stability. Using NMR spectroscopy, small-angle X-ray light scattering (SAXS), and molecular dynamics simulations, we provide here experimental evidence that ion pairs exist in a SAH in murine myosin 7a (residues 858-935), but that they are not fixed or long lasting. In silico modeling revealed that the ion pairs within this α-helix exhibit dynamic behavior, rapidly forming and breaking and alternating between different partner residues. The low-energy helical state was compatible with a great variety of ion pair combinations. Flexible ion pair formation utilizing a subset of those available at any one time avoided the entropic penalty of fixing side chain conformations, which likely contributed to helix stability overall. These results indicate the dynamic nature of ion pairs in SAHs. More broadly, thermodynamic stability in other proteins is likely to benefit from the dynamic behavior of multi-option solvent-exposed ion pairs.

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.

Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability.

Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Thus, we designed and tested simple de novo 98-residue polypeptides containing 7-residue repeats (AEEEXXX, where X is K or R) expected to promote salt-bridge formation between Glu and Lys/Arg. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Combining a PDB analysis with molecular modelling provides a rational explanation, demonstrating that Glu and Arg form salt bridges more commonly, utilize a wider range of rotamer conformations, and are more dynamic than Glu-Lys. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in de novo and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized.

Local and macroscopic electrostatic interactions in single α-helices.

The noncovalent forces that stabilize protein structures are not fully understood. One way to address this is to study equilibria between unfolded states and α-helices in peptides. Electrostatic forces-which include interactions between side chains, the backbone and side chains, and side chains and the helix macrodipole-are believed to contribute to these equilibria. Here we probe these interactions experimentally using designed peptides. We find that both terminal backbone-side chain and certain side chain-side chain interactions (which include both local effects between proximal charges and interatomic contacts) contribute much more to helix stability than side chain-helix macrodipole electrostatics, which are believed to operate at larger distances. This has implications for current descriptions of helix stability, the understanding of protein folding and the refinement of force fields for biomolecular modeling and simulations. In addition, this study sheds light on the stability of rod-like structures formed by single α-helices, which are common in natural proteins such as non-muscle myosins.