High Molecular Weight Protein-Like Semiflexible Chains via Bioorthogonal Polymerization of Coiled-Coil Peptides.

Peptides capable of forming homotetrameric coiled-coil bundles are utilized as the monomeric building blocks ("bundlemers") to synthesize protein-like hybrid polymers consisting of covalently linked coiled-coil microdomains with regularly spaced ethylene glycol repeats via step-growth polymerization employing the highly efficient, bioorthogonal tetrazine (Tz) ligation with -cyclooctene (TCO). Polymerization of Tz and TCO-functionalized peptides in aqueous media under strict stoichiometry at Tz or TCO concentrations of 0.1 to 4.

Spiers Memorial Lecture: Recent advances (and challenges) in supramolecular gels.

Supramolecular hydrogels are physical hydrogels that are formed by non-covalent interactions such as hydrogen bonding, electrostatic attraction, hydrophobic interactions, and π-π stacking. Compared to typical, chemically cross-linked hydrogels, supramolecular networks commonly have stimuli-responsive behavior including reversibility and injectability, which are being widely studied for uses in drug delivery, tissue engineering, and wound healing. This review highlights recent developments in supramolecular network design and behavior focusing on the different possible molecular building blocks, including peptides, polysaccharides, synthetic polymers, and multicomponent systems.

Toward understanding biomolecular materials comprising intrinsically disordered proteins simulation and experiment.

Intrinsically disordered proteins (IDPs) yield solutions with tunable phase transition behavior and have been widely applied in designing stimuli-responsive materials. Understanding interactions between amino acid residues of the IDP sequence is critical to designing new IDP-based materials with selective phase behavior, assembly, and mechanical properties. The lack of defined structure for this class of proteins complicates accurate prediction of their molecular-scale behavior.

Nonequilibrium Solution-Based Assemblies from Bottlebrush Block Copolymers for Drug Delivery.

Kinetic aspects of the self-assembly process of block copolymers are of great interest, as they can direct assembly through specific pathways, yielding nonequilibrium states with complex and unprecedented nanostructures. Assembly kinetics of diblock bottlebrushes was shown to influence the material properties of their solid-state nanostructures, yet little is known regarding their solution-based structures. Herein, we target the nonequilibrium self-assembly of nanoparticles from a zwitterionic diblock bottlebrush consisting of poly(d,l-lactide) and poly(2-methacryloyloxyethyl phosphorylcholine) side-chains.

Architectural control of rod-coil block polypeptide thermoresponsive self-assembly design of coiled-coil orientation.

The architectural control of the self-assembly of a series of block polypeptides comprising a concatenation of an elastin-like peptide and a coiled-coil, bundle-forming peptide (ELP-BFPs), has been demonstrated. Assembly of the polypeptides is controlled by coacervation of the hydrophobic ELP domain, while the type of coiled-coil assembly of the BFP and the specific placement of short histidine tags significantly tunes assembly behavior. Spectrophotometric analysis of self-assembly demonstrated that the transition temperature of assembly can be controlled by the design of the BFP domain and positioning of the His-tags in the constructs.

Sequential Growth of Quantized Peptide Brushes on Colloidal Gold.

We synthesized rigid, macromolecular brushes with well-defined and quantized brush lengths on a gold nanoparticle substrate by using a macromolecular "grafting from" approach. The macromonomers used in these brushes were thiol- and maleimide-functionalized peptide coiled coil "bundlemers" that fold into discrete 4 nm × 2 nm (length × diameter) cylindrical nanoparticles. With each added peptide macromonomer layer, brush thickness increased by approximately the length of a single bundlemer nanoparticle.

Complementary Peptide Interactions Support the Ultra-Rigidity of Polymers of De Novo Designed Click-Functionalized Bundlemers.

Computationally designed 29-residue peptides yield tetra-α-helical bundles with symmetry. The "bundlemers" can be bifunctionally linked via thiol-maleimide cross-links at their N-termini, yielding supramolecular polymers with unusually large, micrometer-scale persistence lengths. To provide a molecularly resolved understanding of these systems, all-atom molecular modeling and simulations of linked bundlemers in explicit solvent are presented.

Ordered assemblies of peptide nanoparticles with only positive charge.

Surface charge patchiness of different charge types can influence the solution behaviours of colloidal particles and globular proteins. Herein, coiled-coil 'bundlemer' nanoparticles that display only a single type of surface charge (SC) are computationally designed to compare their solution behaviours to mixed charge-type (MC) counterparts with both positively and negatively charged side chains. Nematic and columnar liquid crystal phases are discovered in low concentrations of SC particles, indicative of particle end-to-end stacking into columns combined with lateral electrostatic repulsion between columns, while MC particles with the same net charge and particle shape produced only amorphous, soluble aggregates.

Liquid Crystal Behavior of Uniform Short Rods Made from Computationally Designed Parallel Coiled Coil Building Blocks.

Parallel, homotetrameric coiled coils were computationally designed using 29 amino acid peptides. These parallel coiled coils, called "bundlemers", have symmetry, with all N-termini displayed from one end of the nanoparticle and all C-termini from the opposite end. This anisotropic display of the peptide termini allowed for the functionalization of two sets of nanoparticles with either maleimide or thiol functionality at the N-terminal region of the constituent peptides.

Coarse-Grain Model of Ultrarigid Polymer Rods Comprising Bifunctionally Linked Peptide Bundlemers.

Computationally designed homotetrameric helical peptide bundles have been functionalized at their N-termini to achieve supramolecular polymers, wherein individual bundles ("bundlemers") are the monomeric units. Adjacent bundles are linked via two covalent cross-links. The polymers exhibit a range of conformational properties, including formation of rigid-rods with micrometer-scale persistence lengths.

Ultrastable and Redispersible Zwitterionic Bottlebrush Micelles for Drug Delivery.

Bottlebrush copolymers are increasingly used for drug delivery and biological imaging applications in part due to the enhanced thermodynamic stability of their self-assemblies. Herein, we discuss the effect of hydrophilic block chemistry on the stability of bottlebrush micelles. Amphiphilic bottlebrushes with zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and nonionic polyethylene glycol (PEG) hydrophilic blocks were synthesized by "grafting from" polymerization and self-assembled into well-defined spherical micelles.

Combinatorial design of siloxane-incorporated lipid nanoparticles augments intracellular processing for tissue-specific mRNA therapeutic delivery.

Systemic delivery of messenger RNA (mRNA) for tissue-specific targeting using lipid nanoparticles (LNPs) holds great therapeutic potential. Nevertheless, how the structural characteristics of ionizable lipids (lipidoids) impact their capability to target cells and organs remains unclear. Here we engineered a class of siloxane-based ionizable lipids with varying structures and formulated siloxane-incorporated LNPs (SiLNPs) to control in vivo mRNA delivery to the liver, lung and spleen in mice.

Peptide Bundlemer Networks or Lattices: Controlling Cross-Linking and Self-Assembly Using Protein-like Display of Chemistry.

Coiled-coil 'bundlemer' peptides were selectively modified with allyloxycarbonyl (alloc)-protected lysine, a non-natural amino acid containing an alkene on its side chain. The specific display of this alkene from the coiled-coil surface with protein-like specificity enabled this residue to be used as a covalent linkage for creating peptide networks with controllable properties or as a physical linkage for the self-assembly of bundlemers into unexpected, intricate lattices driven by the hydrophobic nature of the side chain. For network formation, peptides were modified with both alloc-protected lysine and cysteine amino acids for solution assembly into solvent-swollen films and subsequent covalent cross-linking via thiol-ene photo click reactions.

Impact of Peptide Length and Solution Conditions on Tetrameric Coiled Coil Formation.

Unlike naturally derived peptides, computationally designed sequences offer programmed self-assembly and charge display. Herein, new tetrameric, coiled coil-forming peptides were computationally designed ranging from 8 to 29 amino acids in length. Experimental investigations revealed that only the sequences having three or more heptads (i.

Genetically Fused Resilin-like Polypeptide-Coiled Coil Bundlemer Conjugates Exhibit Tunable Multistimuli-Responsiveness and Undergo Nanofibrillar Assembly.

Peptide-based materials are diverse candidates for self-assembly into modularly designed and stimuli-responsive nanostructures with precisely tunable compositions. Here, we genetically fused computationally designed coiled coil-forming peptides to the N- and C-termini of compositionally distinct multistimuli-responsive resilin-like polypeptides (RLPs) of various lengths. The successful expression of these hybrid polypeptides in bacterial hosts was confirmed through techniques such as gel electrophoresis, mass spectrometry, and amino acid analysis.

Computational Prediction of Coiled-Coil Protein Gelation Dynamics and Structure.

Protein hydrogels represent an important and growing biomaterial for a multitude of applications, including diagnostics and drug delivery. We have previously explored the ability to engineer the thermoresponsive supramolecular assembly of coiled-coil proteins into hydrogels with varying gelation properties, where we have defined important parameters in the coiled-coil hydrogel design. Using Rosetta energy scores and Poisson-Boltzmann electrostatic energies, we iterate a computational design strategy to predict the gelation of coiled-coil proteins while simultaneously exploring five new coiled-coil protein hydrogel sequences.

Thermoresponsive Coiled-Coil Peptide-Polymer Grafts.

Alkyl halide side groups are selectively incorporated into monodispersed, computationally designed coiled-coil-forming peptide nanoparticles. Poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) is polymerized from the coiled-coil periphery using photoinitiated atom transfer radical polymerization (photoATRP) to synthesize well-defined, thermoresponsive star copolymer architectures. This facile synthetic route is readily extended to other monomers for a range of new complex star-polymer macromolecules.

Genetic Fusion of Thermoresponsive Polypeptides with UCST-type Behavior Mediates 1D Assembly of Coiled-Coil Bundlemers.

Thermoresponsive resilin-like polypeptides (RLPs) of various lengths were genetically fused to two different computationally designed coiled coil-forming peptides with distinct thermal stability, to develop new strategies to assemble coiled coil peptides via temperature-triggered phase separation of the RLP units. Their successful production in bacterial expression hosts was verified via gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism (CD) spectroscopy, ultraviolet-visible (UV/Vis) turbidimetry, and dynamic light scattering (DLS) measurements confirmed the stability of the coiled coils and showed that the thermosensitive phase behavior of the RLPs was preserved in the genetically fused hybrid polypeptides.

3D Hydrogel Cultures for High-Throughput Drug Discovery.

Our increased understanding of how a cell's microenvironment influences its behavior has fueled an interest in three-dimensional (3D) cell cultures for drug discovery. Particularly, scaffold-based 3D cultures are expected to recapitulate in vivo tissue stiffness and extracellular matrix composition more accurately than standard two-dimensional (2D) monolayer cultures. Here we present a 3D hydrogel cell culture setup suitable for automated screening with standard high-throughput screening (HTS) liquid handling equipment commonly found in a drug discovery laboratory.

Rational Design of Bisphosphonate Lipid-like Materials for mRNA Delivery to the Bone Microenvironment.

The development of lipid nanoparticle (LNP) formulations for targeting the bone microenvironment holds significant potential for nucleic acid therapeutic applications including bone regeneration, cancer, and hematopoietic stem cell therapies. However, therapeutic delivery to bone remains a significant challenge due to several biological barriers, such as low blood flow in bone, blood-bone marrow barriers, and low affinity between drugs and bone minerals, which leads to unfavorable therapeutic dosages in the bone microenvironment. Here, we construct a series of bisphosphonate (BP) lipid-like materials possessing a high affinity for bone minerals, as a means to overcome biological barriers to deliver mRNA therapeutics efficiently to the bone microenvironment .