Vincent P. Conticello

Self-assembly of block copolymers derived from elastin-mimetic polypeptide sequences

Protein polymers derived from elastin-mimetic peptide sequences can be synthesized with near-absolute control of macromolecular architecture using genetic engineering techniques. Elastin-mimetic diblock and triblock copolymers have been prepared using this approach in which the individual elastin blocks display different phase behavior in aqueous solution. The selective collapse of the more hydrophobic blocks above the lower critical solution temperature was employed to drive the thermo-reversible self-assembly of elastin-mimetic diblock and triblock copolymer into protein-based nanoparticles and nano-textured hydrogels, respectively. These materials display considerable promise as biomaterials for applications in drug delivery and soft tissue augmentation.

Smectic ordering in solutions and films of a rod-like polymer owing to monodispersity of chain length.

Solutions and melts of stiff (‘rod-like’) macromolecules often exhibit nematic liquid crystalline phases characterized by orientational, but not positional, molecular order,. Smectic phases, in which macromolecular rods are organized into layers roughly perpendicular to the direction of molecular orientation, are rare, owing at least in part to the polydisperse nature (distribution of chain lengths) of polymers prepared by conventional polymerization processes. Bacterial methods for polypeptide synthesis, in which artificial genes encoding the polymer are expressed in bacterial vectors, offer the opportunity to make macromolecules with very well defined chain lengths. Here we show that a monodisperse derivative of poly(γ-benzyl α,L-glutamate) prepared in this way shows smectic ordering in solution and in films. This result suggests that methods for preparing monodisperse polymers might provide access to new smectic phases with layer spacings that are susceptible to precise control on the scale of tens of nanometres.

Atomic-accuracy models from 4.5-A cryo-electron microscopy data with density-guided iterative local refinement

We describe a general approach for refining protein structure models on the basis of cryo-electron microscopy maps with near-atomic resolution. The method integrates Monte Carlo sampling with local density-guided optimization, Rosetta all-atom refinement and real-space B-factor fitting. In tests on experimental maps of three different systems with 4.5-Å resolution or better, the method consistently produced models with atomic-level accuracy largely independently of starting-model quality, and it outperformed the molecular dynamics–based MDFF method. Cross-validated model quality statistics correlated with model accuracy over the three test systems.

Thermoplastic Elastomer Hydrogels via Self‐Assembly of an Elastin‐Mimetic Triblock Polypeptide

Synthetic polymers consisting of well-defined blocks of compositionally dissimilar monomers undergo microscopic phase separation in the solid state and in selective solvents to afford ordered microstructures that display unique, technologically significant properties in comparison to blends of the respective homopolymers. [1] However, the synthetic repertoire of these materials has been limited to tapered blocks of uniform sequence, which potentially restricts the functional complexity of the resulting microstructures. Genetic engineering of synthetic polypeptides enables preparation of block copolymers composed of complex sequences in which the individual blocks may have different mechanical, chemical, or biological properties. [2‐6] The segregation of the protein blocks into compositionally, structurally, and spatially distinct domains should occur in analogy with synthetic block copolymers, affording ordered structures on the nanometer to micrometer size range. The utility of these protein materials depends on the ability to functionally emulate or enhance the materials properties of conventional polymer systems, while retaining the benefits of greater control over the sequence and microstructure that protein engineering affords for the construction of materials. We report herein the genetically directed synthesis and characterization of a triblock copolymer 1 that is derived from an elastin-mimetic polypeptide sequence in which the respective blocks exhibit different mechanical properties (Scheme 1). Moreover, the sequences of the respective blocks were chosen such that the polypeptide undergoes reversible microscopic phase separation from aqueous solution to form a thermoplastic elastomer hydrogel above a lower critical solution temperature Tt.

Elastin-Mimetic Protein Polymers Capable of Physical and Chemical Crosslinking

We report the synthesis of a new class of recombinant elastin-mimetic triblock copolymer capable of both physical and chemical crosslinking. These investigations were motivated by a desire to capture features unique to both physical and chemical crosslinking schemes so as to exert optimal control over a wide range of potential properties afforded by protein-based multiblock materials. We postulated that by chemically locking a multiblock protein assembly in place, functional responses that are linked to specific domain structures and morphologies may be preserved over a broader range of loading conditions that would otherwise disrupt microphase structure solely stabilized by physical crosslinking. Specifically, elastic modulus was enhanced and creep strain reduced through the addition of chemical crosslinking sites. Additionally, we have demonstrated excellent in vivo biocompatibility of glutaraldehyde treated multiblock systems.

Structurally Defined Nanoscale Sheets from Self-Assembly of Collagen-Mimetic Peptides

We report the design of two collagen-mimetic peptide sequences, NSI and NSII, that self-assemble into structurally defined nanoscale sheets. The underlying structure of these nanosheets can be understood in terms of the layered packing of collagen triple helices in two dimensions. These nanosheet assemblies represent a novel morphology for collagen-based materials, which, on the basis of their defined structure, may be envisioned as potentially biocompatible platforms for controlled presentation of chemical functionality at the nanoscale. The molecularly programmed self-assembly of peptides NSI and NSII into nanosheets suggests that sequence-specific macromolecules offer significant promise as design elements for two-dimensional (2D) assemblies. This investigation provides a design rubric for fabrication of structurally defined, peptide-based nanosheets using the principles of solution-based self-assembly facilitated through complementary electrostatic interactions.

Rational Design of Helical Nanotubes from Self-assembly of Coiled-coil Lock Washers

Design of a structurally defined helical assembly is described that involves recoding of the amino acid sequence of peptide GCN4-pAA. In solution and the crystalline state, GCN4-pAA adopts a 7-helix bundle structure that resembles a supramolecular lock washer. Structurally informed mutagenesis of the sequence of GCN4-pAA afforded peptide 7HSAP1, which undergoes self-association into a nanotube via noncovalent interactions between complementary interfaces of the coiled-coil lock-washer structures. Biophysical measurements conducted in solution and the solid state over multiple length scales of structural hierarchy are consistent with self-assembly of nanotube structures derived from 7-helix bundle subunits. The dimensions of the supramolecular assemblies are similar to those observed in the crystal structure of GCN4-pAA. Fluorescence studies of the interaction of 7HSAP1 with the solvatochromic fluorophore PRODAN indicated that the nanotubes could encapsulate shape-appropriate small molecules with high binding affinity.

Cotranslational Incorporation of a Structurally Diverse Series of Proline Analogues in an Escherichia coli Expression System

A set of Escherichia coli expression strains have been defined that are competent for the incorporation of a structurally diverse series of proline analogues under culture conditions that are compatible with high levels of analogue substitution within a proline‐rich protein substrate. These bacterial strains have been employed to assay the efficacy of incorporation of noncanonical amino acids into a recombinant‐protein test substrate and to create variant polypeptides in which native protein sequences have been globally substituted with imino acid analogues in response to proline codons. We envision that these methods may be used to interrogate the effect of imino acid substitution on protein structure and function and may be particularly informative in the context of structural comparison of a series of modified proteins with respect to the stereoelectronic differences between the incorporated proline analogues.

Deformation Responses of a Physically Cross-Linked High Molecular Weight Elastin-Like Protein Polymer

Recombinant protein polymers were synthesized and examined under various loading conditions to assess the mechanical stability and deformation responses of physically cross-linked, hydrated, protein polymer networks designed as triblock copolymers with central elastomeric and flanking plastic-like blocks. Uniaxial stress-strain properties, creep and stress relaxation behavior, as well as the effect of various mechanical preconditioning protocols on these responses were characterized. Significantly, we demonstrate for the first time that ABA triblock protein copolymers when redesigned with substantially larger endblock segments can withstand significantly greater loads. Furthermore, the presence of three distinct phases of deformation behavior was revealed upon subjecting physically cross-linked protein networks to step and cyclic loading protocols in which the magnitude of the imposed stress was incrementally increased over time. We speculate that these phases correspond to the stretch of polypeptide bonds, the conformational changes of polypeptide chains, and the disruption of physical cross-links. The capacity to select a genetically engineered protein polymer that is suitable for its intended application requires an appreciation of its viscoelastic characteristics and the capacity of both molecular structure and conditioning protocols to influence these properties.

Structural Plasticity of Helical Nanotubes Based on Coiled-Coil Assemblies

Numerous instances can be seen in evolution in which protein quaternary structures have diverged while the sequences of the building blocks have remained fairly conserved. However, the path through which such divergence has taken place is usually not known. We have designed two synthetic 29-residue α-helical peptides, based on the coiled-coil structural motif, that spontaneously self-assemble into helical nanotubes in vitro. Using electron cryomicroscopy with a newly available direct electron detection capability, we can achieve near-atomic resolution of these thin structures. We show how conservative changes of only one or two amino acids result in dramatic changes in quaternary structure, in which the assemblies can be switched between two very different forms. This system provides a framework for understanding how small sequence changes in evolution can translate into very large changes in supramolecular structure, a phenomenon that may have significant implications for the de novo design of synthetic peptide assemblies.