Navigating the structural landscape of de Novo α-helical bundles

The association of amphipathic α helices in water leads to α-helical-bundle protein structures. However, the driving force for thisthe hydrophobic effectis not specific and does not define the number or the orientation of helices in the associated state. Rather, this is achieved through deeper sequence-to-structure relationships, which are increasingly being discerned. For example, for one structurally extreme but nevertheless ubiquitous class of bundlethe α-helical coiled coilsrelationships have been established that discriminate between all-parallel dimers, trimers, and tetramers. Association states above this are known, as are antiparallel and mixed arrangements of the helices. However, these alternative states are less well understood. Here, we describe a synthetic-peptide system that switches between parallel hexamers and various up–down–up–down tetramers in response to single-amino-acid changes and solution conditions. The main accessible states of each peptide variant are characterized fully in solution and, in most cases, to high resolution with X-ray crystal structures. Analysis and inspection of these structures helps rationalize the different states formed. This navigation of the structural landscape of α-helical coiled coils above the dimers and trimers that dominate in nature has allowed us to design rationally a well-defined and hyperstable antiparallel coiled-coil tetramer (apCC-Tet). This robust de novo protein provides another scaffold for further structural and functional designs in protein engineering and synthetic biology

Structural resolution of switchable states of a de novo peptide assembly

De novo protein design is advancing rapidly. However, most designs are for single states. Here we report a de novo designed peptide that forms multiple α-helical-bundle states that are accessible and interconvertible under the same conditions. Usually in such designs amphipathic α helices associate to form compact structures with consolidated hydrophobic cores. However, recent rational and computational designs have delivered open α-helical barrels with functionalisable cavities. By placing glycine judiciously in the helical interfaces of an α-helical barrel, we obtain both open and compact states in a single protein crystal. Molecular dynamics simulations indicate a free-energy landscape with multiple and interconverting states. Together, these findings suggest a frustrated system in which steric interactions that maintain the open barrel and the hydrophobic effect that drives complete collapse are traded-off. Indeed, addition of a hydrophobic co-solvent that can bind within the barrel affects the switch between the states both in silico and experimentally

Coiled coils 9-to-5: rational de novo design of α-helical barrels with tunable oligomeric states

The rational design of linear peptides that assemble controllably and predictably in water is challenging. Short sequences must encode unique target structures and avoid alternative states. However, the non-covalent forces that stabilize and discriminate between states are weak. Nonetheless, for α-helical coiled-coil assemblies considerable progress has been made in rational de novo design. In these, sequence repeats of nominally hydrophobic (h) and polar (p) residues, hpphppp, direct the assembly of amphipathic helices into dimeric to tetrameric bundles. Expanding this pattern to hpphhph can produce larger α-helical barrels. Here, we show that pentameric to nonameric barrels are accessed by varying the residue at one of the h sites. In peptides with four L/I–K–E–I–A–x–Z repeats, decreasing the size of Z from threonine to serine to alanine to glycine gives progressively larger oligomers. X-ray crystal structures of the resulting α-helical barrels rationalize this: side chains at Z point directly into the helical interfaces, and smaller residues allow closer helix contacts and larger assemblies

How coiled-coil assemblies accommodate multiple aromatic residues

Rational protein design requires understanding the contribution of each amino acid to a targeted protein fold. For a subset of protein structures, namely, α-helical coiled coils (CCs), knowledge is sufficiently advanced to allow the rational de novo design of many structures, including entirely new protein folds. Current CC design rules center on using aliphatic hydrophobic residues predominantly to drive the folding and assembly of amphipathic α helices. The consequences of using aromatic residues—which would be useful for introducing structural probes, and binding and catalytic functionalities—into these interfaces are not understood. There are specific examples of designed CCs containing such aromatic residues, e.g., phenylalanine-rich sequences, and the use of polar aromatic residues to make buried hydrogen-bond networks. However, it is not known generally if sequences rich in tyrosine can form CCs, or what CC assemblies these would lead to. Here, we explore tyrosine-rich sequences in a general CC-forming background and resolve new CC structures. In one of these, an antiparallel tetramer, the tyrosine residues are solvent accessible and pack at the interface between the core and the surface. In another more complex structure, the residues are buried and form an extended hydrogen-bond network

Differential sensing with arrays of de novo designed peptide assemblies

Differential sensing attempts to mimic the mammalian senses of smell and taste to identify analytes and complex mixtures. In place of hundreds of complex, membrane-bound G-protein coupled receptors, differential sensors employ arrays of small molecules. Here we show that arrays of computationally designed de novo peptides provide alternative synthetic receptors for differential sensing. We use self-assembling α-helical barrels (αHBs) with central channels that can be altered predictably to vary their sizes, shapes and chemistries. The channels accommodate environment-sensitive dyes that fluoresce upon binding. Challenging arrays of dye-loaded barrels with analytes causes differential fluorophore displacement. The resulting fluorimetric fingerprints are used to train machine-learning models that relate the patterns to the analytes. We show that this system discriminates between a range of biomolecules, drink, and diagnostically relevant biological samples. As αHBs are robust and chemically diverse, the system has potential to sense many analytes in various settings

Selective coordination of three transition metal ions within a coiled-coil peptide scaffold

Designing peptides that fold and assemble in response to metal ions tests our understanding of how peptide folding and metal binding influence one another. Here, histidine residues are introduced into the hydrophobic core of a coiled-coil trimer, generating a peptide that self-assembles upon the addition of metal ions. HisAD, the resulting peptide, is unstructured in the absence of metal and folds selectively to form an α-helical construct upon complexation with Cu(II) and Ni(II) but not Co(II) or Zn(II). The structure, and metal-binding ability, of HisAD is probed using a combination of circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. These show the peptide is trimeric and binds to both Cu(II) and Ni(II) in a 1 : 1 ratio with the histidine residues involved in the metal coordination, as designed. The X-ray crystal structure of the HisAD-Cu(II) complex reveals the trimeric HisAD peptide coordinates three Cu(II) ions; this is the first example of such a structure. Additionally, HisAD demonstrates an unprecedented discrimination between transition metal ions, the basis of which is likely to be related to the stability of the peptide-metal complexes formed.The work was supported by the NWO via a VENI grant (722.015.006) to ALB. MR acknowledges funding from the European Union’s Horizon 2020 research and innovation programme through a Marie Skłodowska-Curie Grant (Agreement No. 705857). A VICI grant (724.014.001) awarded to AK funded NSAC. GGR is supported by a European Research Council Advanced Grant (340764). NSP acknowledges the support of an NWO ZonMW grant (91116025). NS acknowledges grant number BIO2015-64216-P (MINECO/AEI/FEDER/UE)

De novo designed peptides for cellular delivery and subcellular localisation

Increasingly, it is possible to design peptide and protein assemblies de novo from first principles or computationally. This approach provides new routes to functional synthetic polypeptides, including designs to target and bind proteins of interest. Much of this work has been developed in vitro. Therefore, a challenge is to deliver de novo polypeptides efficiently to sites of action within cells. Here we describe the design, characterisation, intracellular delivery, and subcellular localisation of a de novo synthetic peptide system. This system comprises a dual-function basic peptide, programmed both for cell penetration and target binding, and a complementary acidic peptide that can be fused to proteins of interest and introduced into cells using synthetic DNA. The designs are characterised in vitro using biophysical methods and X-ray crystallography. The utility of the system for delivery into mammalian cells and subcellular targeting is demonstrated by marking organelles and actively engaging functional protein complexes

<i>De Novo</i>-Designed α‑Helical Barrels as Receptors for Small Molecules

We describe <i>de novo</i>-designed α-helical barrels (αHBs) that bind and discriminate between lipophilic biologically active molecules. αHBs have five or more α-helices arranged around central hydrophobic channels the diameters of which scale with oligomer state. We show that pentameric, hexameric, and heptameric αHBs bind the environmentally sensitive dye 1,6-diphenylhexatriene (DPH) in the micromolar range and fluoresce. Displacement of the dye is used to report the binding of nonfluorescent molecules: palmitic acid and retinol bind to all three αHBs with submicromolar inhibitor constants; farnesol binds the hexamer and heptamer; but β-carotene binds only the heptamer. A co-crystal structure of the hexamer with farnesol reveals oriented binding in the center of the hydrophobic channel. Charged side chains engineered into the lumen of the heptamer facilitate binding of polar ligands: a glutamate variant binds a cationic variant of DPH, and introducing lysine allows binding of the biosynthetically important farnesol diphosphate