Competition between ß-Sheet and Coacervate Domains Yields Diverse Morphologies in Mixtures of Oppositely Charged Homochiral Polypeptides.

Biomolecular assembly processes involving competition between specific intermolecular interactions and thermodynamic phase instability have been implicated in a number of pathological states and technological applications of biomaterials. As a model for such processes, aqueous mixtures of oppositely charged homochiral polypeptides such as poly-l-lysine and poly-l-glutamic acid have been reported to form either ß-sheet-rich solid-like precipitates or liquid-like coacervate droplets depending on competing hydrogen bonding interactions. Herein, we report studies of polypeptide mixtures that reveal unexpectedly diverse morphologies ranging from partially coalescing and aggregated droplets to bulk precipitates, as well as a previously unreported re-entrant liquid-liquid phase separation at high polypeptide concentration and ionic strength. Combining our experimental results with all-atom molecular dynamics simulations of folded polypeptide complexes reveals a concentration dependence of ß-sheet-rich secondary structure, whose relative composition correlates with the observed macroscale morphologies of the mixtures. These results elucidate a crucial balance of interactions that are important for controlling morphology during coacervation in these and potentially similar biologically relevant systems.

Tailoring Writability and Performance of Star Block Copolypeptides Hydrogels through Side-Chain Design.

Shear-recoverable hydrogels based on block copolypeptides with rapid self-recovery hold potential in extrudable and injectable 3D-printing applications. In this work, a series of 3-arm star-shaped block copolypeptides composed of an inner hydrophilic poly(l-glutamate) domain and an outer ß-sheet forming domain is synthesized with varying side chains and block lengths. By changing the ß-sheet forming domains, hydrogels with diverse microstructures and mechanical properties are prepared and structure-function relationships are determined using scattering and rheological techniques. Differences in the properties of these materials are amplified during direct-ink writing with a strong correlation observed between printability and material chemistry. Significantly, it is observed that non-canonical ß-sheet blocks based on phenyl glycine form more stable networks with superior mechanical properties and writability compared to widely used natural amino acid counterparts. The versatile design available through block copolypeptide materials provides a robust platform to access tunable material properties based solely on molecular design. These systems can be exploited in extrusion-based applications such as 3D-printing without the need for additives.

Modular Synthesis and Patterning of High-Stiffness Networks by Postpolymerization Functionalization with Iron-Catechol Complexes.

Bioinspired iron-catechol cross-links have shown remarkable success in increasing the mechanical properties of polymer networks, in part due to clustering of Fe3+-catechol domains which act as secondary network reinforcing sites. We report a versatile synthetic procedure to prepare modular PEG-acrylate networks with independently tunable covalent bis(acrylate) and supramolecular Fe3+-catechol cross-linking. Initial control of network structure is achieved through radical polymerization and cross-linking, followed by postpolymerization incorporation of catechol units via quantitative active ester chemistry and subsequent complexation with iron salts. By tuning the ratio of each building block, dual cross-linked networks reinforced by clustered iron-catechol domains are prepared and exhibit a wide range of properties (Young's moduli up to ∼245 MPa), well beyond the values achieved through purely covalent cross-linking. This stepwise approach to mixed covalent and metal-ligand cross-linked networks also permits local patterning of PEG-based films through masking techniques forming distinct hard, soft, and gradient regions.

Rational mechanochemical design of Diels-Alder crosslinked biocompatible hydrogels with enhanced properties.

An important but often overlooked feature of Diels-Alder (DA) cycloadditions is the ability for DA adducts to undergo mechanically induced cycloreversion when placed under force. Herein, we demonstrate that the commonly employed DA cycloaddition between furan and maleimide to crosslink hydrogels results in slow gelation kinetics and "mechanolabile" crosslinks that relate to reduced material strength. Through rational computational design, "mechanoresistant" DA adducts were identified by constrained geometries simulate external force models and employed to enhance failure strength of crosslinked hydrogels. Additionally, utilization of a cyclopentadiene derivative, spiro[2.4]hepta-4,6-diene, provided mechanoresistant DA adducts and rapid gelation in minutes at room temperature. This study illustrates that strategic molecular-level design of DA crosslinks can provide biocompatible materials with improved processing, mechanical durability, lifetime, and utility.

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.

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.

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.