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 β-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.
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 β-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 β-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.
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.
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.
Bundlemer (grey cylinders) solution structure at various concentrations (C) and length scales (Q-1) is dictated by short-range attraction (green) and long-range repulsion (yellow) interactions.
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•Bundlemers are model globular protein mimicking colloidal particles.•Bundlemers can be computationally desinged to display desired net charge and pattern.•Bundlemers display a balance of attraction and repulsion interactions.
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.
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 β‐sheet forming domain is synthesized with varying side chains and block lengths. By changing the β‐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 β‐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.
A robust strategy to create additive‐free 3D‐printable biomaterials based on β‐sheet–driven hydrogelation of 3‐arm star block copolypeptides that are readily synthesized using N‐carboxyanhydride ring‐opening polymerization is described. The yielding and strain recovery trends correlate with microstructure, filament uniformity, and overall shape fidelity, providing biocompatible polymers using amino‐acid building blocks in tunable hydrogels.
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.
Exact display of side chains using computational design of expressed bundlemer-forming peptides.
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•Computationally designed coiled coil bundlemers are protein-mimicking modular ...building blocks for targeted self-assembly.•Recombinant expression of designed peptides in Escherichia Coli enables production of large quantities of bundlemers.•Resulting bundlemers can be used to self-assemble biomaterials for both biological and non-biological applications.
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.
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.
Coiled-coil peptides have proven useful in a range of materials applications ranging from the formation of well-defined fibrils to responsive hydrogels. The ability to design from first principles ...their oligomerization and subsequent higher order assembly offers their expanded use in producing new materials. Toward these ends, homo-tetrameric, antiparallel, coiled-coil, peptide bundles have been designed computationally, synthesized via solid-phase methods, and their solution behavior characterized. Two different bundle-forming peptides were designed and examined. Within the targeted coiled coil structure, both bundles contained the same hydrophobic core residues. However, different exterior residues on the two different designs yielded sequences with different distributions of charged residues and two different expected isoelectric points of pI 4.4 and pI 10.5. Both coiled-coil bundles were extremely stable with respect to temperature (Tm > 80 C) and remained soluble in solution even at high (millimolar) peptide concentrations. The coiled-coil tetramer was confirmed to be the dominant species in solution by analytical sedimentation studies and by small-angle neutron scattering, where the scattering form factor is well represented by a cylinder model with the dimensions of the targeted coiled coil. At high concentrations (5-15 mM), evidence of interbundle structure was observed via neutron scattering. At these concentrations, the synthetic bundles form soluble aggregates, and interbundle distances can be determined via a structure factor fit to scattering data. The data support the successful design of robust coiled-coil bundles. Despite their different sequences, each sequence forms loosely associated but soluble aggregates of the bundles, suggesting similar dissociated states for each. The behavior of the dispersed bundles is similar to that observed for natural proteins.
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