Tijana Z. Grove

Ligand binding by repeat proteins: natural and designed.

Repeat proteins contain tandem arrays of small structural motifs. As a consequence of this architecture, they adopt non-globular, extended structures that present large, highly specific surfaces for ligand binding. Here we discuss recent advances toward understanding the functional role of this unique modular architecture. We showcase specific examples of natural repeat proteins interacting with diverse ligands and also present examples of designed repeat protein-ligand interactions.

A modular approach to the design of protein‐based smart gels

The modular nature of repeat proteins makes them a versatile platform for the design of smart materials with predetermined properties. Here, we present a general strategy for combining protein modules with specified stability and function into arrays for the assembly of stimuli‐responsive gels. We have designed tetratricopeptide repeat (TPR) arrays which contain peptide‐binding modules that specify the strength and reversibility of network crosslinking in combination with spacer modules that specify crosslinking geometry and overall stability of the array. By combining such arrays with multivalent peptide ligands, self‐supporting stimuli‐responsive gels are formed. Using microrheology, we characterized the kinetics of gelation as a function of concentration and stoichiometry of the components. We also show that such gels are effective in encapsulating and releasing small molecules. Moreover, TPR gels alone are fully compatible with cell growth, whereas gels loaded with an anticancer compound release the compound, resulting in cell death. Thus, we have demonstrated that this new class of tunable biomaterials is ripe for further development as tissue engineering and drug delivery platform.

New materials from proteins and peptides.

In this review we highlight recent accomplishments in the design of materials from proteins and peptides. Examples include hydrogels made from aggregating designed β-hairpin peptides, whose physical properties respond to small changes in the amino acid composition of the peptide; materials that combine different segments of natural elastomeric proteins - such as elastin, resilin, silk fibroin whose bulk properties are dictated in unanticipated ways by their composition; and hydrogels formed by strings or arrays of protein modules, which are cross-linked by multivalent versions of their peptide ligands, and which may exhibit exquisite stimuli-responsive behavior. The suitability of the unique properties of such new materials for practical applications is also considered.

Nanostructured functional films from engineered repeat proteins

Fundamental advances in biotechnology, medicine, environment, electronics and energy require methods for precise control of spatial organization at the nanoscale. Assemblies that rely on highly specific biomolecular interactions are an attractive approach to form materials that display novel and useful properties. Here, we report on assembly of films from the designed, rod-shaped, superhelical, consensus tetratricopeptide repeat protein (CTPR). We have designed three peptide-binding sites into the 18 repeat CTPR to allow for further specific and non-covalent functionalization of films through binding of fluorescein labelled peptides. The fluorescence signal from the peptide ligand bound to the protein in the solid film is anisotropic, demonstrating that CTPR films can impose order on otherwise isotropic moieties. Circular dichroism measurements show that the individual protein molecules retain their secondary structure in the film, and X-ray scattering, birefringence and atomic force microscopy experiments confirm macroscopic alignment of CTPR molecules within the film. This work opens the door to the generation of innovative biomaterials with tailored structure and function.

Consensus design of a NOD receptor leucine rich repeat domain with binding affinity for a muramyl dipeptide, a bacterial cell wall fragment.

Repeat proteins have recently emerged as especially well‐suited alternative binding scaffolds due to their modular architecture and biophysical properties. Here we present the design of a scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Consensus sequence design has emerged as a protein design tool to create de novo proteins that capture sequence‐structure relationships and interactions present in nature. The multiple sequence alignment of 311 individual LRRs, which are the putative ligand‐recognition domain in NOD proteins, resulted in a consensus sequence protein containing two internal and N‐ and C‐capping repeats named CLRR2. CLRR2 protein is a stable, monomeric, and cysteine free scaffold that without any affinity maturation displays micromolar binding to muramyl dipeptide, a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand.

Repeat-Proteins Films Exhibit Hierarchical Anisotropic Mechanical Properties

Complex hierarchical structures provide beneficial structure-property relationships that can be exploited for a variety of applications in engineering and biomedical fields. Here we report on molecular organization and resulting mechanical properties of self-assembled designed repeat-protein films. Wide-angle X-ray diffraction indicates the designed 18-repeat concensus tetratricopeptide repeat protein (CTPR18) orients normal to the casting surface, while small-angle measurements and electron microscopy show a through-plane transversely aligned laminar sheet-like morphology. Self-assembly is driven by the combination of CTPRs head-to-tail stacking and weak dipole-dipole interactions. We highlight the effect that this hierarchical structure has on the materials mechanical properties. We use nanoindentation and dynamic mechanical analysis to test the mechanical properties over multiple length scales, from the molecular level to the bulk. We find that morphology predictably affects the films mechanics from the nano- to the macroscale, with the axial modulus values ranging from 2 to 5 GPa. The predictable nature of the structure-property relationship of CTPR proteins and their assemblies proves them a promising platform for material engineering.

Protein-aided formation of triangular silver nanoprisms with enhanced SERS performance

In this work, we present a modified seed-mediated synthetic strategy for the growth of silver nanoprisms with low shape polydispersity, narrow size distribution and tailored plasmonic absorbance. During the seed nucleation step, consensus sequence tetratricopeptide repeat (CTPR) proteins are utilized as potent stabilizers to facilitate the formation of planar-twinned Ag seeds. Ag nanoprisms were produced in high yield in a growth solution containing ascorbic acid and CTPR-stabilized Ag seeds. From the time-course UV-Vis and transmission electron microscopy (TEM) studies, we postulate that the growth mechanism is the combination of facet selective lateral growth and thermodynamically driven Ostwald ripening. The resultant Ag nanotriangles (NTs) exhibit excellent surface enhanced Raman spectroscopy (SERS) performance. The enhancement factor (EF) measured for the 4-mercapto benzoic acid (4-MBA) reporter is estimated to be 3.37 × 105 in solution and 2.8 × 106 for the SERS substrate.

Repeat protein mediated synthesis of gold nanoparticles: effect of protein shape on the morphological and optical properties

Repeat proteins have recently emerged as promising candidates for the modular design of biohybrid platforms with a high degree of tunability. Consensus sequence tetratricopetide repeat (CTPR) proteins with increasing number of repeats were designed to probe the effects of protein shape on the morphology and resulting physicochemical properties of plasmonic gold nanoparticles. In a synthetic procedure analogous to the biomineralization processes in nature, CTPRs with 3, 6, or 18 tandem repeats were used as both the stabilizing and shape-directing agent. The electronic microscopy and spectroscopic studies indicate that both the [HAuCl4]/[CTPR] ratio and the CTPR shape have dramatic implications on the morphology and plasmon absorbance of the as-synthesized Au NPs. Induced plasmon ellipticity and fluorescence quenching data provide further evidence for the molecular interaction between CTPR and Au NPs or HAuCl4 species. Overall, this work elucidated the effects of CTPR protein shape on the morphology and plasmonic properties of Au NPs, which will further guide the rational design of modular protein based bioconjugate frameworks for colorimetric and enantiomeric biosensors.

Facile, tunable, and SERS-enhanced HEPES gold nanostars

Gold nanoparticles have shown promise as effective tools in biomedicine for drug delivery, imaging, and photo thermal therapy. We present the preparation of gold nanostars (AuNSs) through a two-step approach utilizing a common Goods buffer, HEPES, as a weak reducing agent. AuNSs with tunable branch-lengths up to 30 nm in length were produced in a two-step synthesis. We have investigated the effect of the ionic strength on the AuNS growth and branch length. AuNSs were found to be near-infrared responsive and provide an augmented SERS signal, with an enhancement factor, defined as the magnitude of SERS signal amplification, of approximately 107. The unique branched morphology of the AuNSs confers optical properties advantageous for in vivo biomedical applications.

Surface grafting of chitosan shell, polycaprolactone core fiber meshes to confer bioactivity

Electrospinning of polyesters (e.g. polycaprolactone) is an attractive approach for fabricating meshes with mechanical properties suitable for orthopedic tissue engineering applications. However, the resultant fused-fiber meshes are biologically inert, necessitating surface grafting of bioactive factors to stimulate cell adhesion. In this study, hydrophilic meshes displaying primary amine groups were prepared by coaxially electrospinning fibers with a chitosan/poly(ethylene oxide) shell and a polycaprolactone core. These chitosan–polycaprolactone fiber meshes were mechanically robust (Young’s modulus of 10.1 ± 1.6 MPa under aqueous conditions) with tensile properties comparable to polycaprolactone fiber meshes. Next, the integrin adhesion peptide arginine–glycine–aspartic acid was grafted to chitosan–polycaprolactone fiber meshes. Cell culture studies using bone marrow stromal cells indicated significantly better initial attachment and spreading on arginine–glycine–aspartic acid–conjugated fiber meshes. Specifically, metabolic activity, projected cell area, and cell aspect ratio were all elevated relative to cells seeded on polycaprolactone and unmodified chitosan–polycaprolactone meshes. These results demonstrate a flexible two-step process for creating bioactive electrospun fiber meshes.