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.e., 21 or more amino acids) exhibited coiled coil behavior. The shortest stable coiled coil sequence had a melting temperature (Tm) of approximately 58 ± 1 °C, making it ideal for thermoreversible assembly over moderate temperatures. Effects of pH and monovalent salt were examined, revealing structural stability over a pH range of 4 to 11 and an enhancement in Tm with the addition of salt. The incorporation of the coiled coil as a hydrogel cross-linker results in a thermally and mechanically reversible hydrogel. A subsequent demonstration of the hydrogel printed through a syringe illustrated one of many potential uses from 3D printing to injectable hydrogel drug delivery.

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. Circular dichroism spectroscopy and ultraviolet-visible turbidimetry demonstrated that despite the fusion of disparate structural and responsive units, the coiled coils remained stable in the hybrid polypeptides, and the sequence-encoded differences in thermoresponsive phase separation of the RLPs were preserved. Cryogenic transmission electron microscopy and coarse-grained modeling showed that after thermal annealing in solution, the hybrid polypeptides adopted a closed loop conformation and assembled into nanofibrils capable of further hierarchically organizing into cluster structures and ribbon-like structures mediated by the self-association tendency of the RLPs.

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. Provided this library, we explore the impact of in silico energies on structure and gelation kinetics, where we also reveal a range of blue autofluorescence that enables hydrogel disassembly and recovery. As a result of this library, we identify the new coiled-coil hydrogel sequence, Q5, capable of gelation within 24 h at 4 °C, a more than 2-fold increase over that of our previous iteration Q2. The fast gelation time of Q5 enables the assessment of structural transition in real time using small-angle X-ray scattering (SAXS) that is correlated to coarse-grained and atomistic molecular dynamics simulations revealing the supramolecular assembling behavior of coiled-coils toward nanofiber assembly and gelation. This work represents the first system of hydrogels with predictable self-assembly, autofluorescent capability, and a molecular model of coiled-coil fiber formation.

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. Cryogenic-transmission electron microscopy and coarse-grained modeling revealed that functionalizing the coiled coils with thermoresponsive RLPs leads to their thermally triggered noncovalent assembly into nanofibrillar assemblies.

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. Further, we describe the steps required to validate the assay system for compound screening.

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 in vivo. Following in vitro screening of BP lipid-like materials formulated into LNPs, we identified a lead BP-LNP formulation, 490BP-C14, with enhanced mRNA expression and localization in the bone microenvironment of mice in vivo compared to 490-C14 LNPs in the absence of BPs. Moreover, BP-LNPs enhanced mRNA delivery and secretion of therapeutic bone morphogenetic protein-2 from the bone microenvironment upon intravenous administration. These results demonstrate the potential of BP-LNPs for delivery to the bone microenvironment, which could potentially be utilized for a range of mRNA therapeutic applications including regenerative medicine, protein replacement, and gene editing therapies.

Computational Design of Homotetrameric Peptide Bundle Variants Spanning a Wide Range of Charge States.

With the ability to design their sequences and structures, peptides can be engineered to realize a wide variety of functionalities and structures. Herein, computational design was used to identify a set of 17 peptides having a wide range of putative charge states but the same tetrameric coiled-coil bundle structure. Calculations were performed to identify suitable locations for ionizable residues (D, E, K, and R) at the bundle's exterior sites, while interior hydrophobic interactions were retained. The designed bundle structures spanned putative charge states of -32 to +32 in units of electron charge. The peptides were experimentally investigated using spectroscopic and scattering techniques. Thermal stabilities of the bundles were investigated using circular dichroism. Molecular dynamics simulations assessed structural fluctuations within the bundles. The cylindrical peptide bundles, 4 nm long by 2 nm in diameter, were covalently linked to form rigid, micron-scale polymers and characterized using transmission electron microscopy. The designed suite of sequences provides a set of readily realized nanometer-scale structures of tunable charge that can also be polymerized to yield rigid-rod polyelectrolytes.

Computational Design of Single-Peptide Nanocages with Nanoparticle Templating.

Protein complexes perform a diversity of functions in natural biological systems. While computational protein design has enabled the development of symmetric protein complexes with spherical shapes and hollow interiors, the individual subunits often comprise large proteins. Peptides have also been applied to self-assembly, and it is of interest to explore such short sequences as building blocks of large, designed complexes. Coiled-coil peptides are promising subunits as they have a symmetric structure that can undergo further assembly. Here, an α-helical 29-residue peptide that forms a tetrameric coiled coil was computationally designed to assemble into a spherical cage that is approximately 9 nm in diameter and presents an interior cavity. The assembly comprises 48 copies of the designed peptide sequence. The design strategy allowed breaking the side chain conformational symmetry within the peptide dimer that formed the building block (asymmetric unit) of the cage. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques showed that one of the seven designed peptide candidates assembled into individual nanocages of the size and shape. The stability of assembled nanocages was found to be sensitive to the assembly pathway and final solution conditions (pH and ionic strength). The nanocages templated the growth of size-specific Au nanoparticles. The computational design serves to illustrate the possibility of designing target assemblies with pre-determined specific dimensions using short, modular coiled-coil forming peptide sequences.

Colloid-like solution behavior of computationally designed coiled coil bundlemers.

The use of isotropic potential models of simple colloids for describing complex protein-protein interactions is a topic of ongoing debate in the biophysical community. This contention stems from the unavailability of synthetic protein-like model particles that are amenable to systematic experimental characterization. In this article, we test the utility of colloidal theory to capture the solution structure, interactions and dynamics of novel globular protein-mimicking, computationally designed peptide assemblies called bundlemers that are programmable model systems at the intersection of colloids and proteins. Small-angle neutron scattering (SANS) measurements of semi-dilute bundlemer solutions in low and high ionic strength solution indicate that bundlemers interact locally via repulsive interactions that can be described by a screened repulsive potential. We also present neutron spin echo (NSE) spectroscopy results that show high-Q freely-diffusive dynamics of bundlemers. Importantly, formation of clusters due to short-range attractive, inter-bundlemer interactions is observed in SANS even at dilute bundlemer concentrations, which is indicative of the complexity of the bundlemer charged surface. The similarities and differences between bundlemers and simple colloidal as well as complex protein-protein interactions is discussed in detail.

Peptide Design and Self-assembly into Targeted Nanostructure and Functional Materials.

Peptides have been extensively utilized to construct nanomaterials that display targeted structure through hierarchical assembly. The self-assembly of both rationally designed peptides derived from naturally occurring domains in proteins as well as intuitively or computationally designed peptides that form ß-sheets and helical secondary structures have been widely successful in constructing nanoscale morphologies with well-defined 1-d, 2-d, and 3-d architectures. In this review, we discuss these successes of peptide self-assembly, especially in the context of designing hierarchical materials. In particular, we emphasize the differences in the level of peptide design as an indicator of complexity within the targeted self-assembled materials and highlight future avenues for scientific and technological advances in this field.

One-Component Multifunctional Sequence-Defined Ionizable Amphiphilic Janus Dendrimer Delivery Systems for mRNA.

Efficient viral or nonviral delivery of nucleic acids is the key step of genetic nanomedicine. Both viral and synthetic vectors have been successfully employed for genetic delivery with recent examples being DNA, adenoviral, and mRNA-based Covid-19 vaccines. Viral vectors can be target specific and very efficient but can also mediate severe immune response, cell toxicity, and mutations. Four-component lipid nanoparticles (LNPs) containing ionizable lipids, phospholipids, cholesterol for mechanical properties, and PEG-conjugated lipid for stability represent the current leading nonviral vectors for mRNA. However, the segregation of the neutral ionizable lipid as droplets in the core of the LNP, the "PEG dilemma", and the stability at only very low temperatures limit their efficiency. Here, we report the development of a one-component multifunctional ionizable amphiphilic Janus dendrimer (IAJD) delivery system for mRNA that exhibits high activity at a low concentration of ionizable amines organized in a sequence-defined arrangement. Six libraries containing 54 sequence-defined IAJDs were synthesized by an accelerated modular-orthogonal methodology and coassembled with mRNA into dendrimersome nanoparticles (DNPs) by a simple injection method rather than by the complex microfluidic technology often used for LNPs. Forty four (81%) showed activity in vitro and 31 (57%) in vivo. Some, exhibiting organ specificity, are stable at 5 °C and demonstrated higher transfection efficiency than positive control experiments in vitro and in vivo. Aside from practical applications, this proof of concept will help elucidate the mechanisms of packaging and release of mRNA from DNPs as a function of ionizable amine concentration, their sequence, and constitutional isomerism of IAJDs.

Nanofibers Produced by Electrospinning of Ultrarigid Polymer Rods Made from Designed Peptide Bundlemers.

Mimicking the hierarchical assembly of natural fiber materials is an important design challenge in the manufacturing of nanostructured materials with biomolecules such as peptides. Here, we produce nanofibers with control of structure over multiple length scales, ranging from peptide molecule assembly into supramolecular building blocks called "bundlemers," to rigid-rod formation through a covalent connection of bundlemer building blocks, and, ultimately, to uniaxially oriented fibers made with the rigid-rod polymers. The peptides are designed to physically assemble into coiled-coil bundles, or bundlemers, and to covalently interact in an end-to-end fashion to produce the rigid-rod polymer. The resultant rodlike polymer exhibits a rigid, cylindrical nanostructure confirmed by transmission electron microscopy (TEM) and, correspondingly, exhibits shear-thinning behavior at low shear rates observed in many nanoscopic rod systems. The rigid-rod chains are further organized into final fiber materials via electrospinning processing, all the while preserving their unique rodlike structural characteristics. Morphological and structural investigations of the nanofibers through scanning electron microscopy, transmission electron microscopy, and X-ray scattering, as well as molecular characterization via Fourier transform infrared (FTIR) and Raman spectroscopy, show that continuous nanofibers are composed of oriented rigid-rod chains constituted by α-helical peptides within bundle building blocks. Mechanical properties of electrospun fibers are also presented. The ability to produce nanofibers from the oriented rigid-rod polymer reveals bundlemer chains as a viable tool for the development of new fiber materials with targeted structure and properties.

Recombinant expression of computationally designed peptide-bundlemers in Escherichia coli.

Computational design of fully artificial peptides is extensively researched by material scientists and engineers for the construction of novel nanostructures and biomaterials. Such design has yielded a peptide-based building block or bundlemer, a coiled coil peptide assembly that undergoes further physical-covalent interactions to form 1D, 2D and, potentially, 3D hierarchical assemblies and displays targeted and biomimetic material properties. Recombinant expression is a convenient, flexible tool to synthesize such artificial and modified peptides in large quantities while also enabling economical synthesis of isotopically labeled peptides and longer protein-like artificial peptides. This report describes the protocol for recombinant expression of a 31-amino acid, computationally designed bundlemer-forming peptide in Escherichia coli. Peptide yields of 10 mgs per liter of media were achieved which highlights complementary advantages of recombinant expression technique relative to conventional laboratory-scale synthesis, such as solid-phase peptide synthesis.

Polymers with controlled assembly and rigidity made with click-functional peptide bundles.

The engineering of biological molecules is a key concept in the design of highly functional, sophisticated soft materials. Biomolecules exhibit a wide range of functions and structures, including chemical recognition (of enzyme substrates or adhesive ligands1, for instance), exquisite nanostructures (composed of peptides2, proteins3 or nucleic acids4), and unusual mechanical properties (such as silk-like strength3, stiffness5, viscoelasticity6 and resiliency7). Here we combine the computational design of physical (noncovalent) interactions with pathway-dependent, hierarchical 'click' covalent assembly to produce hybrid synthetic peptide-based polymers. The nanometre-scale monomeric units of these polymers are homotetrameric, α-helical bundles of low-molecular-weight peptides. These bundled monomers, or 'bundlemers', can be designed to provide complete control of the stability, size and spatial display of chemical functionalities. The protein-like structure of the bundle allows precise positioning of covalent linkages between the ends of distinct bundlemers, resulting in polymers with interesting and controllable physical characteristics, such as rigid rods, semiflexible or kinked chains, and thermally responsive hydrogel networks. Chain stiffness can be controlled by varying only the linkage. Furthermore, by controlling the amino acid sequence along the bundlemer periphery, we use specific amino acid side chains, including non-natural 'click' chemistry functionalities, to conjugate moieties into a desired pattern, enabling the creation of a wide variety of hybrid nanomaterials.

Polyelectrolyte character of rigid rod peptide bundlemer chains constructed via hierarchical self-assembly.

Short α-helical peptides were computationally designed to self-assemble into robust coiled coils that are antiparallel, homotetrameric bundles. These peptide bundle units, or 'bundlemers', have been utilized as anisotropic building blocks to construct bundlemer-based polymers via a hierarchical, hybrid physical-covalent assembly pathway. The bundlemer chains were constructed using short linker connections via 'click' chemistry reactions between the N-termini of bundlemer constituent peptides. The resulting bundlemer chains appear as extremely rigid, cylindrical rods in transmission electron microscopy (TEM) images. Small angle neutron scattering (SANS) shows that these bundlemer chains exist as individual rods in solution with a cross-section that is equal to that of a single coiled coil bundlemer building block of ≈20 Å. SANS further confirms that the interparticle solution structure of the rigid rod bundlemer chains is heterogeneous and responsive to solution conditions, such as ionic-strength and pH. Due to their peptidic constitution, the bundlemer assemblies behave like polyelectrolytes that carry an average charge density of approximately 3 charges per bundlemer as determined from SANS structure factor data fitting, which describes the repulsion between charged rods in solution. This repulsion manifests as a correlation hole in the scattering profile that is suppressed by dilution or addition of salt. Presence of rod cluster aggregates with a mass fractal dimension of ≈2.5 is also confirmed across all samples. The formation of such dense, fractal-like cluster aggregates in a solution of net repulsive rods is a unique example of the subtle balance between short-range attraction and long-rage repulsion interactions in proteins and other biomaterials. With computational control of constituent peptide sequences, it is further possible to deconvolute the underlying sequence driven structure-property relationships in the modular bundlemer chains.

Computational Reverse-Engineering Analysis for Scattering Experiments on Amphiphilic Block Polymer Solutions.

In this paper, we present a computational reverse-engineering analysis for scattering experiments (CREASE) based on genetic algorithms and molecular simulation to analyze the structure within self-assembled amphiphilic polymer solutions. For a given input comprised of scattering intensity profiles and information about the amphiphilic polymers in solution, CREASE outputs the structure of the self-assembled micelles (e.g., core and corona diameters, aggregation number) as well as the conformations of the amphiphilic polymer chains in the micelle (e.g., blocks' radii of gyration, chain radii of gyration, monomer concentration profiles). First, we demonstrate CREASE's ability to reverse-engineer self-assembled nanostructures for scattering profiles obtained from molecular simulations (or in silico experiments) of generic coarse-grained bead-spring polymer chains in an implicit solvent. We then present CREASE's outputs for scattering profiles obtained from small-angle neutron scattering (SANS) experiments of poly(d-glucose carbonate) block copolymers in solution that exhibit assembly into spherical nanoparticles. The success of this method is demonstrated by its ability to replicate, quantitatively, the results from in silico experiments and by the agreement in micelle core and corona sizes obtained from microscopy of the in vitro solutions. The primary strength of CREASE is its ability to analyze scattering profiles without an off-the-shelf scattering model and the ability to provide chain and monomer level structural information that is otherwise difficult to obtain from scattering and microscopy alone.

The peptide hormone glucagon forms amyloid fibrils with two coexisting ß-strand conformations.

Glucagon and insulin maintain blood glucose homeostasis and are used to treat hypoglycemia and hyperglycemia, respectively, in patients with diabetes. Whereas insulin is stable for weeks in its solution formulation, glucagon fibrillizes rapidly at the acidic pH required for solubility and is therefore formulated as a lyophilized powder that is reconstituted in an acidic solution immediately before use. Here we use solid-state NMR to determine the atomic-resolution structure of fibrils of synthetic human glucagon grown at pharmaceutically relevant low pH. Unexpectedly, two sets of chemical shifts are observed, indicating the coexistence of two ß-strand conformations. The two conformations have distinct water accessibilities and intermolecular contacts, indicating that they alternate and hydrogen bond in an antiparallel fashion along the fibril axis. Two antiparallel ß-sheets assemble with symmetric homodimer cross sections. This amyloid structure is stabilized by numerous aromatic, cation-π, polar and hydrophobic interactions, suggesting mutagenesis approaches to inhibit fibrillization could improve this important drug.

Implementation of a High-Throughput Pilot Screen in Peptide Hydrogel-Based Three-Dimensional Cell Cultures.

Cell-based high-throughput drug screening (HTS) is a common starting point for the drug discovery and development process. Currently, there is a push to combine complex cell culture systems with HTS to provide more clinically applicable results. However, there are mechanistic requirements inherent to HTS as well as material limitations that make this integration challenging. Here, we used the peptide-based shear-thinning hydrogel MAX8 tagged with the RGDS sequence to create a synthetic extracellular scaffold to culture cells in three dimensions and showed a preliminary implementation of the scaffold within an automated HTS setup using a pilot drug screen targeting medulloblastoma, a pediatric brain cancer. A total of 2202 compounds were screened in the 384-well format against cells encapsulated in the hydrogel as well as cells growing on traditional two-dimensional (2D) plastic. Eighty-two compounds passed the first round of screening at a single point of concentration. Sixteen-point dose-response was done on those 82 compounds, of which 17 compounds were validated. Three-dimensional (3D) cell-based HTS could be a powerful screening tool that allows researchers to finely tune the cell microenvironment, getting more clinically applicable data as a result. Here, we have shown the successful integration of a peptide-based hydrogel into the high-throughput format.