Raymond S. Tu

Engineering of an environmentally responsive beta roll peptide for use as a calcium-dependent cross-linking domain for peptide hydrogel formation.

We have created a set of rationally designed peptides that form calcium-dependent hydrogels based on the beta roll peptide domain. In the absence of calcium, the beta roll domain is intrinsically disordered. Upon the addition of calcium, the peptide forms a beta helix secondary structure. We have designed two variations of our beta roll domain. First, we have mutated one face of the beta roll domain to contain leucine residues so that the calcium-dependent structural formation leads to dimerization through hydrophobic interactions. Second, an α-helical leucine zipper domain is appended to the engineered beta roll domain as an additional means of forming intermolecular cross-links. This full peptide construct forms a hydrogel only in calcium-rich environments. The resulting structural and mechanical properties of the supramolecular assemblies are compared with the wild-type domain using several biophysical techniques including circular dichroism, FRET, bis-ANS binding and microrheology. The calcium responsiveness and rheological properties of the leucine beta roll containing construct confirm the potential of this allosterically regulated scaffold to serve as a cross-linking domain for stimulus-responsive biomaterials development.

Compositional control of higher order assembly using synthetic collagen peptides.

We present the case of a two-component collagen peptide hydrogel that self-assembles through noncovalent electrostatic interactions. Natural collagen materials, such as those of connective tissue or the basement membrane, assemble in a hierarchic fashion. Similarly, the synthetic peptides presented here proceed from monomer to trimer to fiber and, finally, to a hydrogel. By varying stoichiometry and concentration, we are able to dissect the stages of higher order assembly. Insight gained from this study will improve the molecular design of biomimetic materials.

Cooperative DNA binding and assembly by a bZip peptide-amphiphile

The bipartite basic zipper (bZip) GCN4 peptide, containing a leucine zipper and a basic binding region, is a well-studied transcription factor that can be rationally adapted to control dimerization or assembly. We have covalently appended alkyl tails to the C-terminus (leucine zipper terminus) of a bZip sequence, yielding mono- and dialkyl bZip peptide-amphiphiles that allowed us to investigate how molecular design can control the formation of secondary structure and self-assembled structure. We demonstrate that these peptide-amphiphiles exhibit four qualities that are representative of their modular construction. First, circular dichroism confirms that self-assembly of peptide-amphiphiles above the critical micelle concentration (CMC) results in an enhanced α-helical secondary structure as peptide head groups are confined to the assembled interface with high local concentrations. Second, the binding of the peptide-amphiphiles to DNA yields a further increase in secondary structure, where the helicity of the basic binding region is stabilized by forming native-like contacts, an “induced fit mechanism”. Third, competitive fluorescence binding assays show peptide-amphiphiles bind cooperatively to DNA well below the CMC, where DNA templates monomeric binding and hydrophobic forces promote cooperativity, but the ability of the peptide to recognize a specific DNA sequence is not retained. And fourth, SANS results demonstrate the assembly of large lamellar aggregates as peptide-amphiphiles complex with DNA, supporting a structural hypothesis in which peptide-amphiphiles bind to the DNA in a native-like ‘standing’ orientation. These designed synthetic molecular architectures are capable of hierarchical assembly making them useful as functional building blocks that may be applied to a variety of systems, including gene delivery and artificial transcription factors.

Collapse of Particle-Laden Interfaces under Compression: Buckling vs Particle Expulsion

Colloidal particles can bind to fluid interfaces with a capillary energy that is thousands of times the thermal energy. This phenomenon offers an effective route to emulsion and foam stabilization where the stability is influenced by the phase behavior of the particle-laden interface under deformation. Despite the vast interest in particle-laden interfaces, the key factors that determine the collapse of such an interface under compression have remained relatively unexplored. In this study, we illustrate the significance of the particle surface wettability and presence of electrolyte in the subphase on interparticle interactions at the interface and the resulting collapse mode. Various collapse mechanisms including buckling, particle expulsion, and multilayer formation are reported and interpreted in terms of particle-particle and particle-interface interactions.

Bionanocomposites: differential effects of cellulose nanocrystals on protein diblock copolymers.

We investigate the effects of mixing a colloidal suspension of tunicate-derived cellulose nanocrystals (t-CNCs) with aqueous colloidal suspensions of two protein diblock copolymers, EC and CE, which bear two different self-assembling domains (SADs) derived from elastin (E) and the coiled-coil region of cartilage oligomeric matrix protein (C). The resulting aqueous mixtures reveal improved mechanical integrity for the CE+t-CNC mixture, which exhibits an elastic gel network. This is in contrast to EC+t-CNC, which does not form a gel, indicating that block orientation influences the ability to interact with t-CNCs. Surface analysis and interfacial characterization indicate that the differential mechanical properties of the two samples are due to the prevalent display of the E domain by CE, which interacts more with t-CNCs leading to a stronger network with t-CNCs. On the other hand, EC, which is predominantly C-rich on its surface, does not interact as much with t-CNCs. This suggests that the surface characteristics of the protein polymers, due to folding and self-assembly, are important factors for the interactions with t-CNCs, and a significant influence on the overall mechanical properties. These results have interesting implications for the understanding of cellulose hydrophobic interactions, natural biomaterials and the development of artificially assembled bionanocomposites.

Mechanical Stability of Polystyrene and Janus Particle Monolayers at the Air/Water Interface

The compressional instability of particle-laden air/water interfaces is investigated with plain and surface-anisotropic (Janus) particles. We hypothesize that the amphiphilic nature of Janus particles leads to both anisotropic particle-particle and particle-interface interactions that can yield particle films with unique collapse mechanisms. Analysis of Langmuir isotherms and microstructural characterization of the homogeneous polystyrene particle films during compression reveal an interfacial buckling instability followed by folding, which is in good agreement with predictions from classical elasticity theory. In contrast, Janus particle films exhibit a different behavior during compression, where the collapse mode occurs through the subduction of the Janus particle film. Our results suggest that particle-laden films comprised of surface-anisotropic particles can be engineered to evolve new material properties.

Modulating Supramolecular Assemblies and Mechanical Properties of Engineered Protein Materials by Fluorinated Amino Acids

Here we describe the biosynthesis and characterization of fluorinated protein block polymers comprised of the two self-assembling domains (SADs): elastin (E) and the coiled-coil region of cartilage oligomeric matrix proteins (C). Fluorination is achieved by residue-specific incorporation of p-fluorophenylalanine (pFF) to create pFF-EC, pFF-CE, and pFF-ECE. Global fluorination results in downstream effects on the temperature-dependent secondary structure, supramolecular assembly, and bulk mechanical properties. The impact of fluorination on material properties also differs depending on the orientation of the block configurations as well as the number of domains in the fusion. These studies suggest that integration of fluorinated amino acids within protein materials can be employed to tune the material properties, especially mechanical integrity.

Supramolecular assembly and small molecule recognition by genetically engineered protein block polymers composed of two SADs

Genetically engineered protein block polymers are an important class of biomaterials that have gained significant attention in recent years due to their potential applications in biotechnology, electronics and medicine. The majority of the protein materials have been composed of at least a single self-assembling domain (SAD), enabling the formation of supramolecular structures. Recently, we developed block polymers consisting of two distinct SADs derived from an elastin-mimetic polypeptide (E) and the alpha-helical COMPcc (C). These protein polymers, synthesized as the block sequences--EC, CE, and ECE--were assessed for overall conformation and macroscopic thermoresponsive behavior. Here, we investigate the supramolecular assembly as well as the small molecule binding and release profile of these block polymers. Our results demonstrate that the protein polymers assemble into particles as well as fully or partially networked structures in a concentration dependent manner that is distinct from the individual E and C homopolymers and the E+C non-covalent mixture. In contrast to synthetic block polymers, the structured assembly, binding and release abilities are highly dependent on the composition and orientation of the blocks. These results reveal the promise for these block polymers for therapeutic delivery and biomedical scaffolds.

Artificial protein block polymer libraries bearing two SADs: effects of elastin domain repeats.

We have generated protein block polymer E(n)C and CE(n) libraries composed of two different self-assembling domains (SADs) derived from elastin (E) and the cartilage oligomeric matrix protein coiled-coil (C). As the E domain is shortened, the polymers exhibit an increase in inverse transition temperature (T(t)); however, the range of temperature change differs dramatically between the E(n)C and CE(n) library. Whereas all polymers assemble into nanoparticles, the bulk mechanical properties of the E(n)C are very different from CE(n). The E(n)C members demonstrate viscolelastic behavior under ambient conditions and assemble into elastic soft gels above their T(t) values. By contrast, the CE(n) members are predominantly viscous at all temperatures. All library members demonstrate binding to curcumin. The differential thermoresponsive behaviors of the E(n)C and CE(n) libraries in addition to their small molecule recognition abilities make them suitable for potential use in tissue engineering and drug delivery.

Coupled Folding and Specific Binding: Fishing for Amphiphilicity

Proteins are uniquely capable of identifying targets with unparalleled selectivity, but, in addition to the precision of the binding phenomenon, nature has the ability to find its targets exceptionally quickly. Transcription factors for instance can bind to a specific sequence of nucleic acids from a soup of similar, but not identical DNA strands, on a timescale of seconds. This is only possible with the enhanced kinetics provided for by a natively disordered structure, where protein folding and binding are cooperative processes. The secondary structures of many proteins are disordered under physiological conditions. Subsequently, the disordered structures fold into ordered structures only when they bind to their specific targets. Induced folding of the protein has two key biological advantages. First, flexible unstructured domains can result in an intrinsic plasticity that allows them to accommodate targets of various size and shape. And, second, the dynamics of this folding process can result in enhanced binding kinetics. Several groups have hypothesized the acceleration of binding kinetics is due to induced folding where a “fly-casting” effect has been shown to break the diffusion-limited rate of binding. This review describes experimental results in rationally designed peptide systems where the folding is coupled to amphiphilicity and biomolecular activity.