Engineering repeat proteins of the immune system.

Limitations associated with immunoglobulins have motivated the search for novel binding scaffolds. Repeat proteins have emerged as one promising class of scaffolds, but often are limited to binding protein and peptide targets. An exception is the repeat proteins of the immune system, which have in recent years served as an inspiration for binding scaffolds which can bind glycans and other classes of biomolecule. Like other repeat proteins, these proteins can be very stable and have a monomeric mode of binding, with elongated and highly variable binding surfaces. The ability to target glycans and glycoproteins fill an important gap in current tools for research and biomedical applications.

Protein Self-Assemblies That Can Generate, Hold, and Discharge Electric Potential in Response to Changes in Relative Humidity.

Generation of electric potential upon external stimulus has attracted much attention for the development of highly functional sensors and devices. Herein, we report large-displacement, fast actuation in the self-assembled engineered repeat protein Consensus Tetratricopeptide Repeat protein (CTPR18) materials. The ionic nature of the CTPR18 protein coupled to the long-range alignment upon self-assembly results in the measured conductivity of 7.1 × 10-2 S cm-1, one of the highest reported for protein materials. The change of through-thickness morphological gradient in the self-assembled materials provides the means to select between faster, highly water-sensitive actuation or vastly increased mechanical strength. Tuning of the mode of motion, e.g., bending, twisting, and folding, is achieved by changing the morphological director. We further show that the highly ionic character of CTPR18 gives rise to piezo-like behavior in these materials, exemplified by low-voltage, ionically driven actuation and mechanically driven generation/discharge of voltage. This work contributes to our understanding of the emergence of stimuli-responsiveness in biopolymer assemblies.

Enhanced Fluorescence Properties of Stilbene-Containing Alternating Copolymers.

In recent years, nonconjugated, fluorophore-free organic polymers have emerged as potentially useful light-emitting materials. The fluorescence properties of a novel class of nonconjugated, tert-butyl carboxylate functionalized stilbene-containing alternating copolymers are investigated in this work. These sterically crowded, semi-rigid copolymers exhibit very strong blue fluorescence in organic solvents upon irradiation. The origin of the fluorescent band with high quantum yield is attributed to the "through space" π-π interactions between the phenyl rings from the stilbene and CO groups from the anhydride groups. To the best of our knowledge, the di-tert-butyl group-containing stilbene and maleic anhydride alternating copolymer showed one of the highest fluorescent intensities among all fluorophore-free polymers. The excellent linearity of the luminescence property of this copolymer is an important attribute for future potential quantitative applications. The fluorescence is maintained when the tert-butyl groups are removed and the resulting carboxylic acid-functionalized copolymer is dissolved in water at neutral pH, which can render these copolymers as attractive candidates for diagnostic and therapeutic applications.

Homo- and heteropolymer self-assembly of recombinant trichocytic keratins.

In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues. However, during extraction from natural sources, such as hair and wool fibers, natural keratins are subject to extensive processing conditions that lead to formation of unwanted by-products. Additionally, natural keratins suffer from limited sequence tunability. Recombinant keratin proteins can overcome these drawbacks while maintaining the desired chemical and physical characteristics of natural keratins. Herein, we present the bacterial expression, purification, and solution characterization of human hair keratins K31 and K81. The obligate heterodimerization of the K31/K81 pair that results in formation of intermediate filaments is maintained in the recombinant proteins. Surprisingly, we have for the first time observed new zero- and one-dimensional nanostructures from homooligomerization of K81 and K31, respectively. Further analysis of the self-assembly mechanism highlights the importance of disulfide crosslinking in keratin self-assembly.

Protein Design for Nanostructural Engineering: General Aspects.

This chapter aims to introduce the main challenges in the field of protein design for engineering of nanostructures and functional materials. First, we introduce proteins and illustrate the key characteristics that open many possibilities for the use of proteins in nanotechnology. Then, we describe the current state of the art of nanopatterning techniques and the actual needs of the emerging field of nanotechnology to develop new tools in order to achieve precise control and manipulation of elements at the nanoscale. In this sense, the increasing knowledge of protein science and advances in protein design allow to tackle current challenges such as the design of nanodevices, nanopatterned surfaces, and nanomachines. This book highlights the recent progresses of protein nanotechnology over the last decade and emphasizes the power of protein engineering through illustrative examples of protein based-assemblies and their potential applications.

Protein Design for Nanostructural Engineering: Concluding Remarks and Future Directions.

This final chapter aims to summarize the main conclusions of the book and to point to possible directions for further research in the field of protein design for nanostructural engineering. Even though this research field is still at its infancy, multidisciplinary research efforts in the design of synthetic protein-based nanostructures and functional materials have resulted in significant progress. The chapters in this book cover several selected examples of the most recent advances concerning the use of proteins and peptides as building blocks for the fabrication of architectures and functional nanostructures, assemblies, and materials. Here, we provide a general overview of the strategies that can be employed to prepare functional protein-based nanostructures, and nanostructured materials and devices. Finally, we highlight some of the main aspects to be considered by the research community to set the path for the near future developments.

Design of Self-Assembling Protein-Polymer Conjugates.

Protein-polymer conjugates are of particular interest for nanobiotechnology applications because of the various and complementary roles that each component may play in composite hybrid-materials. This chapter focuses on the design principles and applications of self-assembling protein-polymer conjugate materials. We address the general design methodology, from both synthetic and genetic perspective, conjugation strategies, protein vs. polymer driven self-assembly and finally, emerging applications for conjugate materials. By marrying proteins and polymers into conjugated bio-hybrid materials, materials scientists, chemists, and biologists alike, have at their fingertips a vast toolkit for material design. These inherently hierarchical structures give rise to useful patterning, mechanical and transport properties that may help realize new, more efficient materials for energy generation, catalysis, nanorobots, etc.

Designing repeat proteins for biosensors and medical imaging.

Advances in protein engineering tools, both computational and experimental, has afforded many new protein structures and functions. Here, we present a snapshot of repeat-protein engineering efforts towards new, versatile, alternative binding scaffolds for use in analytical sensors and as imaging agents. Analytical assays, sensors and imaging agents based on the direct binding of analyte are increasingly important for research and diagnostics in medicine, food safety, and national security.

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.

Designed leucine-rich repeat proteins bind two muramyl dipeptide ligands.

Designed protein receptors hold diagnostic and therapeutic promise. We now report the design of five consensus leucine-rich repeat proteins (CLRR4-8) based on the LRR domain of nucleotide-binding oligomerization domain (NOD)-like receptors involved in the innate immune system. The CLRRs bind muramyl dipeptide (MDP), a bacterial cell wall component, with micromolar affinity. The overall Kd app values ranged from 1.0 to 57 µM as measured by fluorescence quenching experiments. Biphasic fluorescence quenching curves were observed in all CLRRs, with higher affinity Kd1 values ranging from 0.04 to 4.5 µM, and lower affinity Kd2 values ranging from 3.1 to 227 µM. These biphasic binding curves, along with the docking studies of MDP binding to CLRR4, suggest that at least two MDPs bind to each protein. Previously, only single MDP binding was reported. This high-capacity binding of MDP promises small, soluble, stable CLRR scaffolds as candidates for the future design of pathogen biosensors.

Stimuli-responsive smart gels realized via modular protein design.

Smart gels have a variety of applications, including tissue engineering and controlled drug delivery. Here we present a modular, bottom-up approach that permits the creation of protein-based smart gels with encoded morphology, functionality, and responsiveness to external stimuli. The properties of these gels are encoded by the proteins from which they are synthesized. In particular, the strength and density of the network of intermolecular cross-links are specified by the interactions of the gels' constituent protein modules with their cognate peptide ligands. Thus, these gels exhibit stimuli-responsive assembly and disassembly, dissolving (or gelling) under conditions that weaken (or strengthen) the protein-peptide interaction. We further demonstrate that such gels can encapsulate and release both proteins and small molecules and that their rheological properties are well suited for biomedical applications.

A comparative study of materials assembled from recombinant K31 and K81 and extracted human hair keratins.

Natural biopolymers have found success in tissue engineering and regenerative medicine applications. Their intrinsic biocompatibility and biological activity make them well suited for biomaterials development. Specifically, keratin-based biomaterials have demonstrated utility in regenerative medicine applications including bone regeneration, wound healing, and nerve regeneration. However, studies of structure-function relationships in keratin biomaterials have been hindered by the lack of homogeneous preparations of materials extracted and isolated from natural sources such as wool and hair fibers. Here we present a side-by-side comparison of natural and recombinant human hair keratin proteins K31 and K81. When combined, the recombinant proteins (i.e. rhK31 and rhK81) assemble into characteristic intermediate filament-like fibers. Coatings made from natural and recombinant dimers were compared side-by-side and investigated for coating characteristics and cell adhesion. In comparison to control substrates, the recombinant keratin materials show a higher propensity for inducing involucrin and hence, maturation in terms of potential skin cell differentiation.

Synthesis of Triangular Silver and Gold Nanoprisms Using Consensus Sequence Tetratricopeptide Repeat Proteins.

Anisotropic metallic nanoparticles, such as Au and Ag nanoprisms (NPSMs), have received tremendous attention for their application in catalysis, molecular sensing, signal amplification, bioimaging, and therapeutic applications due to their shape-dependent optical and physical properties. Herein, we present a protein-enabled synthetic strategy for the seeded growth of silver and gold NPSMs with low shape polydispersity, narrow size distribution, and tailored plasmonic absorbance. During the initial seed nucleation step, consensus sequence tetratricopeptide repeat (CTPR) proteins are utilized as potent stabilizers to facilitate the formation of planar-twinned Ag seeds. High yield production of well-defined Ag/Au NPSMs is achieved, respectively, by adding CTPR-stabilized Ag seeds into the growth solutions containing metal precursor, mild reducing agent, sodium halide, and additional CTPR.

Photo-triggered release of 5-fluorouracil from a MOF drug delivery vehicle.

A nano metal-organic-framework (nanoMOF) was employed as a first-of-its kind drug delivery vehicle (DDV) for the photo-controlled release of therapeutics with simultaneous breakdown of the carrier into small molecules.

Cargo delivery on demand from photodegradable MOF nano-cages.

We report the photo-induced degradation of and cargo release from a nanoscale metal-organic framework (nMOF) incorporating photo-isomerizable 4,4'-azobenzenedicarboxylate (AZB) linkers. The structure matches a UiO-type framework where 12 4,4'-azobenzenedicarboxylate moieties are connected to a Zr6O4(OH)4 cluster, referred to as UiO-AZB. Due to the incorporation of photo-isomerizable struts, the degradation of UiO-AZB is accelerated by irradiation with white light (1.3 ± 0.1% h-1 under dark conditions vs. 8.4 ± 0.4% h-1 when irradiated). Additionally, we show slow release of Nile Red (NR) which is triggered by irradiation (0.04 ± 0.01% h-1 under dark conditions vs. 0.36 ± 0.02% h-1 when irradiated).

Seed-mediated biomineralizaton toward the high yield production of gold nanoprisms.

Gold nanotriangles (Au NTs) with tunable edge length were synthesized via a green chemical route in the presence of the designed consensus sequence tetratricopeptide repeat (CTPR) protein, halide anions (Br(-)) and CTPR-stabilized Ag seeds. The well-defined morphologies, tailored plasmonic absorbance from visible-light to the near infrared (NIR) region, colloidal stability and biocompatibility are attributed to the synergistic action of CTPR, halide ions, and CTPR-stabilized Ag seeds.

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.

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.

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.