Constructing synthetic-protein assemblies from de novo designed 310 helices

Compared with the iconic α helix, 310 helices occur much less frequently in protein structures. The different 310-helical parameters lead to energetically less favourable internal energies, and a reduced tendency to pack into defined higher-order structures. Consequently, in natural proteins, 310 helices rarely extend past 6 residues, and do not form regular supersecondary, tertiary, or quaternary interactions. Here, we show that despite their absence in nature, synthetic protein-like assemblies can be built from 310 helices. We report the rational design, solution-phase characterisation, and an X-ray crystal structure for water-soluble bundles of 310 helices with consolidated hydrophobic cores. The design uses 6-residue repeats informed by analysing natural 310 helices, and incorporates aminoisobutyric acid residues. Design iterations reveal a tipping point between α-helical and 310-helical folding, and identify features required for stabilising assemblies in this unexplored region of protein-structure space.

Solution NMR and racemic crystallography provide insights into a novel structural class of cyclic plant peptides

Head-to-tail cyclic and disulfide-rich peptides are natural products with applications in drug design. Among these are the PawS-Derived Peptides (PDPs) produced in seeds of the daisy plant family. PDP-23 is a unique member of this class in that it is twice the typical size and adopts two β-hairpins separated by a hinge region. The β-hairpins - both stabilised by a single disulfide bond - fold together into a V-shaped tertiary structure creating a hydrophobic core. In water two PDP-23 molecules merge their hydrophobic cores to form a square prism quaternary structure. Here, we synthesised PDP-23 and its enantiomer comprising all D-amino acids, which allowed us to confirm these solution NMR structural data by racemic crystallography. Furthermore, we discovered the related PDP-24. NMR analysis showed that PDP-24 does not form a dimeric structure and it has poor water solubility, but in less polar solvents adopts near identical secondary and tertiary structure to PDP-23. The natural role of these peptides in plants remains enigmatic, as we did not observe any antimicrobial or insecticidal activity. However, the plasticity of these larger PDPs and their ability to change structure under different conditions makes them appealing peptide drug scaffolds.

The OP Protein Cage: A Versatile Molecular Delivery Platform

Well-defined containers constructed from multiple protein subunits are a unique class of nanomaterial useful in supramolecular chemistry and biology. These protein cages are widespread in nature, where they are responsible for a diversity of important tasks. As such, producing our own designer protein cages, complete with bespoke functionalities, is a promising avenue to new nanodevices, biotechnology and therapies. Herein, we describe how an artificial, computationally designed protein cage can be rationally engineered using supramolecular intuition to produce new functional capsules. Positive supercharging the interior cavity of this porous protein cage enables the efficient encapsulation of oligonucleotides by electrostatically-driven self-assembly. Moreover, the resulting cargo-loaded cages enter mammalian cells and release their cargo, for example siRNA which modulates gene expression. To expand the cargo scope of this proteinaceous container, a higher level of supramolecular complexity can also be introduced. Encapsulation of anionic surfactants affords protein-scaffolded micelles, which are capable of sequestering poorly water-soluble small molecules within their hydrophobic cores. These hybrid particles stably carry bioactive cargo and deliver it intracellularly, thereby increasing potency. Further development of these genetically-encoded materials is ongoing towards specific applications ranging from cell biology to medicine.

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.

From Inert Storage to Biological Activity—In Search of Identity for Oxidized Cholesteryl Esters

Esterification of cholesterol is a universal mechanism to store and transport large quantities of cholesterol between organs and tissues and to avoid toxicity of the excess of cellular cholesterol. Intended for transport and storage and thus to be inert, cholesteryl esters (CEs) reside in hydrophobic cores of circulating lipoproteins and intracellular lipid droplets. However, the inert identity of CEs is dramatically changed if cholesterol is esterified to a polyunsaturated fatty acid and subjected to oxidative modification. Post-synthetic, or epilipidomic, oxidative modifications of CEs are mediated by specialized enzymes, chief among them are lipoxygenases, and by free radical oxidation. The complex repertoire of oxidized CE (OxCE) products exhibit various, context-dependent biological activities, surveyed in this review. Oxidized fatty acyl chains in OxCE can be hydrolyzed and re-esterified, thus seeding oxidized moieties into phospholipids (PLs), with OxPLs having different from OxCEs biological activities. Technological advances in mass spectrometry and the development of new anti-OxCE antibodies make it possible to validate the presence and quantify the levels of OxCEs in human atherosclerotic lesions and plasma. The article discusses the prospects of measuring OxCE levels in plasma as a novel biomarker assay to evaluate risk of developing cardiovascular disease and efficacy of treatment.

Alternative Hydrophobic Core in Proteins—The Effect of Specific Synergy

Proteins with a high degree of sequence similarity representing different structures provide a key to understand how protein sequence codes for 3D structure. An analysis using the fuzzy oil drop model was carried out on two pairs of proteins with different secondary structures and with high sequence identities. It has been shown that distributions of hydrophobicity for these proteins are approximated well using single 3D Gaussian function. In other words, the similar sequences fold into different 3D structures, however, alternative structures also have symmetric and monocentric hydrophobic cores. It should be noted that a significant change in the helical to beta-structured form in the N-terminal section takes places in the fragment much preceding the location of the mutated regions. It can be concluded that the final structure is the result of a complicated synergy effect in which the whole chain participates simultaneously.

Amphiphilic Block Copolymers Bearing Hydrophobic γ-Tocopherol Groups with Labile Acetal Bond

High concentrations of γ-tocopherol (γTCP) tend to show antioxidant, anti-inflammatory, and anticancer effects. In this study, we prepared polymer micelles under acidic conditions with a controlled release of γTCP due to the decomposition of pendant acetal bonds. First, a precursor diblock copolymer composed of poly(ethylene glycol) (PEG) and acrylic acid (AA) was prepared. This was followed by the synthesis of an amphiphilic diblock copolymer (PEG54-P(AA/VE6/γTCP29)140), incorporated into hydrophobic γTCP pendant groups attached to the main chain through an acetal bond. The prepared PEG54-P(AA/VE6/γTCP29)140 was further dispersed in water to form polymer micelles composed of hydrophobic cores that were generated from a hydrophobic block containing γTCPs and hydrophilic shells on the surface. Under acidic conditions, γTCP was then released from the core of the polymer micelles due to the decomposition of the pendant acetal bonds. In addition, polymer micelles swelled under acidic conditions due to hydration of the core.

The Amyloid as a Ribbon-Like Micelle in Contrast to Spherical Micelles Represented by Globular Proteins

Selected amyloid structures available in the Protein Data Bank have been subjected to a comparative analysis. Classification is based on the distribution of hydrophobicity in amyloids that differ with respect to sequence, chain length, the distribution of beta folds, protofibril structure, and the arrangement of protofibrils in each superfibril. The study set includes the following amyloids: Aβ (1–42), which is listed as Aβ (15–40) and carries the D23N mutation, and Aβ (11–42) and Aβ (1–40), both of which carry the E22Δ mutation, tau amyloid, and α-synuclein. Based on the fuzzy oil drop model (FOD), we determined that, despite their conformational diversity, all presented amyloids adopt a similar structural pattern that can be described as a ribbon-like micelle. The same model, when applied to globular proteins, results in structures referred to as “globular micelles,” emerging as a result of interactions between the proteins’ constituent residues and the aqueous solvent. Due to their composition, amyloids are unable to attain entropically favorable globular forms and instead attempt to limit contact between hydrophobic residues and water by producing elongated structures. Such structures typically contain quasi hydrophobic cores that stretch along the fibril’s long axis. Similar properties are commonly found in ribbon-like micelles, with alternating bands of high and low hydrophobicity emerging as the fibrils increase in length. Thus, while globular proteins are generally consistent with a 3D Gaussian distribution of hydrophobicity, the distribution instead conforms to a 2D Gaussian distribution in amyloid fibrils.