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.

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.

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.

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.

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.

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.

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.

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.

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 ZrO(OH) 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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

Simultaneous true, gated, and coupled electron-transfer reactions and energetics of protein rearrangement.

Cytochromes c(6) and f react by three et mechanisms under similar conditions. We report temperature and viscosity effects on the protein docking and kinetics of (3)Zncyt c(6)+cyt f(III)→Zncyt c(6)(+)+cyt f(II). At 0.

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.

Creating novel proteins by combining design and selection.

We present the results of combining design and selection to remodel a protein-peptide binding interface, using the peptide PTIEEVD and the TPR1 module interaction as our test case. We initially used the program Rosetta to interrogate possible TPR1 sequences compatible with binding the peptide PTIEEVD. Based on these results, we screened a small library of TPR1 variants, using a split GFP fluorescent assay to identify proteins that are able to bind to the PTIEEVD peptide.

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.