Protein Engineered Triblock Polymers Composed of Two SADs: Enhanced Mechanical Properties and Binding Abilities.

Recombinant methods have been used to engineer artificial protein triblock polymers composed of two different self-assembling domains (SADs) bearing one elastin (E) flanked by two cartilage oligomeric matrix protein coiled-coil (C) domains to generate CEC. To understand how the two C domains improve small molecule recognition and the mechanical integrity of CEC, we have constructed CL44AECL44A, which bears an impaired CL44A domain that is unstructured as a negative control. The CEC triblock polymer demonstrates increased small molecule binding and ideal elastic behavior for hydrogel formation. The negative control CL44AECL44A does not exhibit binding to small molecule and is inelastic at lower temperatures, affirming the favorable role of C domain and its helical conformation. While both CEC and CL44AECL44A assemble into micelles, CEC is more densely packed with C domains on the surface enabling the development of networks leading to hydrogel formation. Such protein engineered triblock copolymers capable of forming robust hydrogels hold tremendous promise for biomedical applications in drug delivery and tissue engineering.

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.

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.

N-terminal aliphatic residues dictate the structure, stability, assembly, and small molecule binding of the coiled-coil region of cartilage oligomeric matrix protein.

The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D(3) (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the alpha-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules.