Protein&#X26;#X02010;Engineered Functional Materials

REVIEW

Engineered Proteins www.advhealthmat.de Protein-Engineered Functional Materials

Yao Wang, Priya Katyal, and Jin Kim Montclare*

temperature, pH, or ionic strength. These Proteins are versatile macromolecules that can perform a variety of functions. triggers can further modulate the design In the past three decades, they have been commonly used as building blocks to and synthesis of novel biomaterials with generate a range of biomaterials. Owing to their flexibility, proteins can either increasingly complex functions. Because of their versatile nature, proteins can be be used alone or in combination with other functional molecules. Advances combined with a variety of other materials in synthetic and chemical biology have enabled new protein fusions as well as to create materials with novel functionali- the integration of new functional groups leading to biomaterials with emergent ties. Additionally, the inherent property of properties. This review discusses protein-engineered materials from the per- proteins to self-assemble has paved the spectives of domain-based designs as well as physical and chemical approaches ways to generate new protein assemblies. By precisely controlling the self-assembly for crosslinked materials, with special emphasis on the creation of hydrogels. of proteins, novel architectures with Engineered proteins that organize or template metal ions, bear noncanonical improved functional properties can be amino acids (NCAAs), and their potential applications, are also reviewed. designed.[3] The increasing interest in self-assembly of proteins has contributed significantly to 1. Introduction the development of biomaterials. These self-assembled protein based materials have a wide range of biomedical applications in Proteins are multifunctional macromolecules that regulate tissue engineering, biosensors, drug delivery, medical imaging, a number of biological processes and pathways.[1,2] With the gene therapies and protein therapeutics.[4–10] The properties of recent advances in protein engineering including synthetic proteins can be dynamically regulated by the organization or and chemical biology, new variants have been designed with templation of inorganic metal ions and more recently by intro- improved or novel functionalities. The diverse properties of ducing noncanonical amino acids (NCAAs).[11,12] proteins make them excellent candidates for building new bio- In this review, we highlight various recombinant proteins materials. Over the past few decades, continuous efforts have with a particular focus on their ability to assemble into various been made to develop protein engineered materials, capable of structures. In the following sections, we discuss functional replacing synthetic polymers, owing to their biocompatible and materials from the perspectives of domain-based designs as biodegradable properties.[1] well as physically and chemically crosslinked materials, with There are several advantages associated with the use of special emphasis on the design of hydrogels. In addition, we protein-based materials. The proteins can undergo conforma- review protein engineered materials bearing sequences for tional changes based on external stimuli, such as changes in metal crystallization and noncanonical amino acids, and their potential applications.

Y. Wang, Dr. P. Katyal, Prof. J. K. Montclare Department of Chemical and Biomolecular Engineering New York University 2. Domain-Based Protein Engineered Materials Tandon School of Engineering Brooklyn, NY 11201, USA 2.1. Single Domain Protein Engineered Materials E-mail: [email protected] Prof. J. K. Montclare Single domain protein engineered materials are comprised of Department of Chemistry sequences that are derived from a single protein conformation. New York University While the sequences of single domain protein may vary with sim- New York, NY 10003, USA ilar or different repeats, the conformation of the domain remains Prof. J. K. Montclare Department of Biomaterials singular. In this section, we describe such single domains that New York University College of Dentistry can interact with each other to form hydrogel networks. New York, NY 10010, USA Prof. J. K. Montclare Department of Radiology 2.1.1. Elastin New York University School of Medicine New York, NY 10016, USA Elastin is a major component of the extracellular matrix The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adhm.201801374. (ECM) that directly impacts the elasticity of blood vessels and overall movement of joints and limbs.[13] Inspired by the DOI: 10.1002/adhm.201801374 natural protein, tailor-made elastin-like polypeptides (ELPs)

Yao Wang was born in Beijing, China, and holds a bachelor’s degree in polymer materials. In 2015, she finished her M.S. in materials science and engineering at the University of Florida. The same year in September, she joined New York University Tandon School of Engineering where she is currently pur- suing her Ph.D. in materials chemistry. Her work focuses on patternable environmentally responsive hydrogels derived from protein tri-block copolymers for biomedical applications.

Priya Katyal completed her Ph.D. in pharmaceutical sciences from the University of Connecticut, where she investigated protein–protein and protein–polymer interac- Figure 1. Plot showing the inverse phase transition of ELPs. Reproduced tions using biophysical and [14] with permission. Copyright 2014, John Wiley and Sons. biochemical approaches. She is a postdoctoral fellow in consisting of pentapeptide sequences have been investigated Professor Montclare’s lab at for their remarkable elasticity and self-assembling properties New York University, Tandon [14] (Figure 1). These properties render ELPs useful for applica- School of Engineering. Her tions in tissue engineering,[15,16] as drug delivery systems,[17–19] research is focused on developing self-assembled inject- and as probes for bioimaging applications.[16,20,21] able hydrogels for post-traumatic osteoarthritis. ELPs are comprised of a pentapeptide sequence, Val-Pro- Gly-Xaa-Gly or (VPGXG) , where ‘Xaa’ is an interchangeable n Jin Kim Montclare is a pro- amino acid (except proline) and “n” is the number of repeating fessor in the Department of units; the properties of ELPs can be tuned by varying Xaa or n Chemical and Biomolecular (Table 1).[16,22] The glycine and proline residues of ELPs main- Engineering at NYU Tandon tain the structure and function of elastin. ELPs exhibit a unique School of Engineering. lower critical solution temperature (LCST), which is also She has appointments referred to as the inverse transition temperature (T ); below t in biochemistry at SUNY their T , ELPs are soluble in aqueous solution and as the tem- t Downstate Medical Center, perature increases beyond T , ELPs undergo phase separation t chemistry at NYU, radiology and form aggregates.[23] Introduction of hydrophobic residues at NYU School of Medicine, at the “Xaa” position decreases T while ionic and polar residues t and biomaterials at NYU increase T . By varying the guest residue, it is possible to tune t College of Dentistry. Professor the ELP transition temperature from 0 to 60 C.[24,25] ° Montclare is performing groundbreaking research in The transition temperature of ELPs is also influenced by engineering proteins to mimic nature and, in some cases, their amino acid sequence, chain length, buffer concentration, work better than nature. She exploits nature’s biosyn- and polypeptide concentration.[26] Chilkoti and co-workers have thetic machinery and evolutionary mechanisms to design designed ELPs that form injectable depots capable of providing new artificial proteins. Her lab focuses on two research sustained release of peptide therapeutics.[22] Additionally, ELP areas: 1) developing protein biomaterials capable of self- solution when mixed with chondrocytes, result in the formation assembling into supramolecular structures and 2) engi- of coacervates that can maintain the cell viability (Figure 2a).[27] neering functional proteins/enzymes for particular The Chilkoti group has precisely tuned the T by varying the t substrates with the aim of targeting human disorders, drug guest residue and ELP chain length (Figure 2b) using recom- delivery and tissue regeneration. binant DNA techniques.[28] They have proposed a model that can predict the Tt of ELPs (Table 1) with the sequence repeats of VPGVG and VPGAG across a range of molecular weights and The Conticello group has generated different types of BAB chain lengths.[28] These studies have allowed the design of new (Table 1) elastin-based triblock protein polymers, capable of ELPs for applications in medicine and biotechnology.[22,26] forming thermoplastic elastomer hydrogels (Figure 3).[29,30]

Table 1. Sequences of single domain proteins.

Type of protein Name of protein Sequence Reference Elastin Protein Elastin Like Proteins (VPGVG)n [24]

(ELPs) (VPGAG)n[(VPGIG)2(VPGKG)(VPGIG)2] [16] [139]

[MSKGPG-(XGVPG)n-Y or WP] [26]

ELP[V5A2G3] -n [28]

ELP[V1A8G7] -n

ELP[V5]-n *n = length of the pentapeptide BAB Thermoplastic Two BAB sequences: [30]

Elastomer Hydrogels VPAVG[(IPAVG)4(VPAVG)]16IPAVG}-[VPGVG[(VPGVG)2VPGEG(VPGVG)2]30VPGVG]{VPAVG[( [29]

IPAVG)4(VPAVG)]16IPAVG}

{VPAVG[(IPAVG)4(VPAVG)]16IPAVG}-[VPGVGVPGVG]-VPGVG{VPAVG[(IPAVG)4(VPAVG)]16 IPAVG} *A block is Bold Hydrophobic B block:

VPAVG[(IPAVG)4(VPAVG)]16IPAVG}-[A]-{VPAVG[(IPAVG)4(VPAVG)]16IPAVG} Hydrophilic elastomeric A blocks:

[VPGVG[(VPGVG)2VPGEG(VPGVG)2]30VPGVG] [VPGVGVPGVG] Resilin Protein Drosophila melanogaster, MVRPEPPVNS YLPPSDSYGA PGQSGPGGRP SDSYGAPGGG NGGRPSDSYG [46] CG15920 gene APGQGQGQGQ GQGGYAGKPS DTYGAPGGGN GNGGRPSSSY GAPGGGNGGR PSDTYGAPGG GNGGRPSDTY GAPGGGGNGN GGRPSSSYGA PGQGQGNGNG GRSSSSYGAP GGGNGGRPSD TYGAPGGGNG GRPSDTYGAP GGGNNGGRPS SSYGAPGGGN GGRPSDTYGA PGGGNGNGSG GRPSSSYGAP GQGQGGFGGR PSDSTGAPGQ NQKPSDSYGA PGSGNGNGGR PSSSYGAPGS GPGGRPSDSY GPPASGSGAG GAGGSGPGGA DYDNDEPAKY EFNYQVEDAP SGLSFGHSEM RDGDFTTGQY NVLLPDGRKQ IVEYEADQQG YRPQIRYEGD ANDGSGPSGP GGPGGQNLGA DHYSSHRPGN GNGNGNGGYS GGRPGGQDLG PSGYSGGRPG GQDLGAGGYS NGKPGGQDLG PGGYSGGRPG GQDLGRDGYS GGRPGGQDLG ASGYGNGRPG GNGNGGSDGG RVIIGGRVIG GQDGGDQGYS GGRPGGQDLG RDGYSSGRPG GRPGGNGQDS QDGQGYSSGR PGQGGRNGFG PGGQNGDNDG SGYRY Exon 1 sequence in italic, Exon 2 sequence in Bold and Exon 3 sequence underlined

Putative Resilin Sequence Signal peptide-(GGRPSDSYGAPGGGN)18-Exon 2-(GYSGGRPGGQDLG)11 [36] from the Drosophila melanogaster CG15290 gene

Rec1-resilin (GGRPSDSYGAPGGGN)17 [34] [41]

Dros16 (GYSGGRPGGQDLG)16 [42]

Two Exons (1 and 3) (GGRPSDSYGAPGGGN)18-(GYSGGRPGGQDLG)11 [46] Anopheles gambiae (AQTPSSQYGAP) [39]

AN16 (AQTPSSQYGAP)16 [43]

Silk Proteins Silkworm Silks from B. mori repeated motifs [GAGSGA]n and [GAGXGA]n [52] X = tyrosine or valine [53]

Spider Silks from N. clavipes (GPGGYGPGQQGPGGYGPGQQGPSGPGS(A)n) [48]

Major Ampullate spidroin 1 SGRGGLGGQAAGAAAAAGGAGQYGGLGSQG)n [57] (MaSp1) Protein Analog [58]

s Helices and COMPcc MGRSH6GSGDLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGKLN [66]

Coiled Coil C MGRSH6-GSIEGRAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTSKL [69]

Proteins Q MGRSH6-GSIEGRVKEITFLKNTAPQMLRELQETNAALQDVRELLRQQSKL [70]

CSP MGRSH6GSGRLRPQMLRELQRTNAALRDVRELLRQQVKEITRLKNTVRRSRASGKLN [71] [72] GCN4-pAA VKQLAMKVEELADANYHLASAVARLANAVGERAKA [74] 7HSAP1 VEKLAQAVEKLARA NEKLAYAVEKLARAVEKLAQA Consensus TPR Consensus TPR Protein WLGYAFAP [76] Protein

Figure 2. a) The process shows how ELP-cell mixtures form coacervates under temperature stimulation and how chondrocytes are captured within the ELP coacervates. Reproduced with permission.[27] Copyright 2002, American Chemical Society. b) Graph shows inverse phase transitions of ELPs with varying length and guest residues. Reproduced with permission.[28] Copyright 2004, American Chemical Society.

The protein copolymer contains a hydrophobic B block derived While the B block governs the Tt; mechanical properties can from the pentapeptide sequence [(V/I)PAVG] and a hydrophilic be tuned by modifying block A.[30] Remarkably, the mechanical elastomeric A block derived from different pentapeptide or tetra- behavior of the copolymer changes from elastomeric to plastic, peptide sequences comprised of [VPXYG], where X is glycine when glycine at the X position is replaced with alanine.[30] The and Y can be changed into glutamic acid that display change unique properties of the thermoplastic hydrogel can be utilized [29–31] [32] in lower critical transition temperature (Tt) (Table 1). for a variety of tissue engineering applications.

Figure 3. Schematic of formation of water-swollen network through micellization of hydrophobic end-block domains (red) of the BAB triblock copolymer . Unassociated unimers in aqueous solution below the phase transition temperature (left) and crosslinked structure between the central hydrophilic (green) and hydrophobic end-block domains (right), respectively. Reproduced with permission.[31] Copyright 2002, Elsevier Science B.V.

Figure 4. a) Structure and reaction of dityrosine formation in resilin. b) Autofluorescence property of resilin pad of the flea in the pleural arch at the top of the hind legs under ultraviolet illumination. Reproduced with permission.[34] Copyright 2005, Nature Publishing Group.

2.1.2. Resilin of recombinant resilin protein derived from the D. melanogaster exon domains (Table 1).[43,44] These repeat motifs are respon- Resilin, an intrinsically disordered protein found in cuticles of sible for the elasticity of resilin. In addition, researchers have winged insects, is known for its elastic properties.[33] In general, engineered recombinant resilin-like polypeptides (RLPs) con- tyrosine residues within the protein sequence react with each taining a number of repeating units based on different species other to form dityrosine and trityrosine, which can further cross- (such as fruit fly, African malaria, flea and mosquitos) and have link to form a network (Figure 4a).[34,35] Owing to its crosslinked also combined them with other functional domains.[36,39] network, natural resilin exhibits high resiliency and a very high Rec1-resilin[34,39,40] (Table 1), derived from the first exon of the fatigue lifetime.[34,36] It is heat stable[33] and demonstrates phase fruit fly gene, exhibit resilience comparable to natural resilin.[45] transition behavior, pH-responsiveness and autofluorescence It exhibits dual upper critical solution temperature (UCST) and properties (Figure 4b).[36,37] These properties make resilin a LCST behavior and can be crosslinked into a rubber like mate- unique biomaterial often referred to as protein rubber.[34,36,37] rial.[37,44] Similar to Rec1-resilin, AN16 (Table 1), derived from Resilin-based materials benefit from its unique LCST phase African malaria mosquito gene Anopheles gambiae (Table 1), [36,46] transition behavior, allowing them to be soluble below their Tt is comprised of 16 copies of proresilin repeats. It has and becoming insoluble with properties similar to natural rub- similar secondary structure and mechanical properties as Rec1- [36] [45] bers above the Tt. These features allow the use of resilin in resilin. AN16 having higher resilience and elasticity, displays biomedical and tissue engineering fields for the design of scaf- UCST at a higher temperature when compared to Rec1-resilin folds, biosensors, and environmentally responsive materials.[38] (Figure 6).[40] Although these RLPs are derived from different Since the discovery of resilin gene in Drosophila melanogaster, sources, they exhibit similar properties as that of natural resilin a flurry of research focused on the construction of resilin-like and can be used as scaffolds for applications including tissue polypeptides has been conducted.[34,39,40] The gene precursor engineering and biosensors.[36] of resilin, gene product CG15920 (Table 1), is comprised of 620 amino acids with a signal peptide sequence at the N-ter- minus and three exon domains (Figure 5).[36] The three exon 2.1.3. Silk and Silk-Like Proteins domains include: the N-terminal elastin domain (exon 1), the chitin-binding domain (exon 2) and the C-terminal elastic Silk is a fibrous protein spun by Lepidoptera larvae such as silk- domain (exon 3).[36] Different copies of the repetitive sequences worms, spiders, scorpions, mites and flies.[47,48] Among these dif- derived from CG15920 gene, particularly from the exon 1 ferent species, silkworm (Bombyx mori) and spider (Nephila clavipes (GGRPSDSYGAPGGGN)n and exon 3 (GYSGGRPGGQDLG)n, and Araneus diadematus) silks are the most extensively character- have been reported.[36] Rec1-resilin[34,41] with 17 copies of the ized with remarkable mechanical properties.[49] 15 repeats of exon 1, Dros16[42] with 16 copies of the 15 repeats Silkworm silks have been first investigated as biomaterials from exon 1 and two exons (1 and 3) are all excellent examples centuries ago and are commonly used as suture materials.[47,48]

Figure 5. The putative resilin sequence from the Drosophila melanogaster CG15290 gene product. It has 620 amino acids and consists of a signal peptide sequence and three main regions. Exon 1 with 18 repeat motifs, exon 2 involved in binding of chitin and exon 3 with 11 repeat motifs. Reproduced with permission.[36] Copyright 2013, Elsevier Ltd.

Silkworm silk from B. mori contains fibroin, the structural protein that forms antiparallel β-sheets, composed of repeated motifs [GAGSGA]n and [GAGXGA]n where X can be tyrosine or valine residues (Table 1).[52,53] Similar to silkworm silk, spider silk from N. clavipes contains glycine and alanine (Table 1). It also includes a high proportion of glutamic acid, proline and arginine.[48] Overall, silk fibroin possesses a higher amino acid composition of glycine and alanine (43% and 30%) than that of spider silk (30% and 20%).[48,54] Both spider and silkworm silks are semicrystalline and selectively incorporate specific repetitive motifs into β-sheet crystallites.[55] The β-sheet conformation help silk proteins maintain their solubility in strong salts and acids during storage and after being spun.[55,56] Kaplan and colleagues have reported that recombinant silk- Figure 6. 3% AN16 solution observed over a range of temperatures for UCST worm silk protein demonstrates relatively weak mechanical behavior. Reproduced with permission.[40] Copyright 2014, Elsevier Ltd. properties and brittle behavior when compared to recombinant spider silk.[57] The spider silk exhibits exceptionally high ten- As recombinant DNA techniques have matured, allowing facile sile strength and remarkable mechanical properties. More spe- cloning and expression through a variety of hosts, rapid devel- cifically, recombinant spider silk protein derived from dragline opment of silk-like proteins ensued.[50] Despite the differences spider silk (Table 1) is soluble in water, organic solvents, and in functionality of silk proteins due to variations in primary ionic liquids and has been used to produce various novel bio- amino acid sequence and processing procedures, they represent materials including fibers and textiles, biofilms, and hydrogels a unique family of structural proteins that are biocompatible, (Figure 7).[48,57] Major ampullate spidroin 1 (MaSp1, Table 1) biodegradable, mechanically superior, and can be chemically protein analog from the N. clavipes spider dragline silk has been modified to suit a wide range of biomedical applications.[47,51] electrospun to form spider silk fibers with diameter ranging

Figure 7. Schematic of recombinant silk processed into new biomaterials. The silk proteins can be solubilized with a variety of solvents, including formic acid, hexafluoroisopropanol, calcium nitrate, lithium salts, or ionic liquids. Once solubilized, the silk protein solutions can be processed into the range of different structures shown. Reproduced with permission.[57] Copyright 2008, Elsevier Ltd.

Adv. Healthcare Mater. 2019, 8, 1801374 1801374 (6 of 33) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advhealthmat.de from 10–60 µm.[57,58] A porous 3D scaffold generated from spider silk fibroin has been used to promote chondrocyte cell growth, with applications in articular cartilage engineering.[57,59]

2.1.4. Engineered Helices and Coiled-Coils

The α-helix is the most common and highly favored stereochem- ical secondary structure present within proteins.[60] Crick and co- workers suggest that the coiled-coil is the predominant structure of the α-class fibrous proteins (keratin, myosin, epidermin, and fibrinogen, also referred as k-m-e-f class).[61]α-helical coiled-coils are ubiquitous in protein–protein interaction domains,[62] found in over 200 natural proteins. These proteins are involved in many biological activities, such as muscle contraction, transcrip- tion, metabolism, membrane channel, molecular chaperons, and immobilization of proteins and enzymes.[60,63] Typically, right-handed α-helices tend to self-assemble into a [64] coiled-coil structure with heptad repeats (abcdefg)n. The e and g positions are often occupied by charged residues (lysine and glutamic acid), while the c, b, and f positions usually contain polar residues. The a and d positions are usually located at the hydrophobic interface of two helices and are mostly nonpolar residues (leucine, isoleucine and valine). Both a and d residue positions determine whether the interhelical interactions between the two coiled-coils would be antiparallel or parallel (Figure 8).[60,62,65] The helices can form a number of oligomers

Figure 9. a) Structure and sequence of COMPcc. Reproduced with permission.[70] Copyright 2014, American Chemical Society. b) Transmission electron micrograph of Q fiber and schematic representation of Q fiber assembly with staggered positive (red) and negative (blue) regions of the pentamer. Reproduced with permission.[71] Copyright 2015, American Chemical Society. c) Schematic of CSP complexation with plasmid DNA and a tertiary complex with cationic lipids to form lipoproteoplexes for gene delivery. Reproduced with permission.[72] Copyright 2014, Elsevier Ltd.

(dimers, tetramers, pentamers, etc.) and attain different topologies to direct a wide variety of protein assemblies.[65] Coiled-coil systems are being studied as models for stability and structural specificity and for their potential as drug delivery vehicles.[64] Montclare and co-workers have investigated α-helical assemblies based on the coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) (Figure 9a, Table 1).[19,66–68] COMPcc self-assembles into a pentameric unit with a hydrophobic pore that can bind small hydrophobic molecules like vitamin D, curcumin and all-trans retinol.[66] They have demonstrated that a s variant of COMPcc, bearing two serines (COMPcc , Table 1) self-assemble into nanofibers; binding to zinc metal ions sta- bilizes the fibers while binding to nickel ions causes aggrega- Figure 8. Top: Antiparallel helical wheel, where residues a and d line up tion.[69] Another variant Q (Table 1), engineered by truncating with d’ and a’, respectively. Bottom: Parallel helical wheel where residues s a and d line up with d’ and a’, respectively. Hydrophobic and ionic inter- the COMPcc by one heptad to form C (Table 1) and swap- actions are highlighted. Reproduced with permission.[65] Copyright 2008, ping regions with respect to glutamine 54, self-assembles into John Wiley & Sons, ltd. nanofibers and upon binding to a small molecule, curcumin,

Adv. Healthcare Mater. 2019, 8, 1801374 1801374 (7 of 33) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advhealthmat.de forms microfibers (Figure 9b).[70,71] Introduction of positively charged residues on the surface of COMPccs to form a super- charged protein, CSP endows it the ability to effectively deliver genes (Table 1).[72] CSP exhibits greater helicity compared to the parent protein and can bind with nucleic acids and cationic lipids to form spherical particles termed “lipoproteoplexes.” These assemblies are capable of condensing and delivering nucleic acids (Figure 9c).[72] CSP has been used as a dual delivery system for delivering siRNA and doxorubicin, a potent anticancer agent, to breast carcinoma MCF7 cells.[73] It has also been used to accelerate wound healing in diabetic mice models.[73] The Conticello group has developed a coiled-coil derived from the leucine zipper region of the Saccharomyces cerevisiae transcrip- tion factor GCN4 to create GCN4-pAA (Table 1).[74] GCN4-pAA self-assembles into a seven-helix (helices A-G) bundle and resem- bles a supramolecular lock-washer as the A and G helices interfaces provide extra surface area for complementary interaction between coiled-coil promoters (Figure 10a). Structurally informed mutagenesis of GCN4-pAA has produced 7HSAP1 (Table 1), which retains the seven-helix bundle and forms nanotubes via noncovalent interactions between the complementary interfaces of the coiled-coil lock-washer structures (Figure 10b,c).[74] These rationally designed helical nanotubes can encapsulate shape- appropriate small molecules such as cyclodextrins with high binding affinity.[74] These examples demonstrate the tremendous technical advantage of coiled-coils as functional nanoporous biomaterials for tissue engineering and drug delivery purposes.[70]

2.1.5. Consensus Tetratricopeptide Repeats Figure 10. a) Helical wheel of GCN4-pAA (left) and seven-helix bundle with helices A–G represented by the different colors (right). Coiled-coil In 1990, both Hirano and Sikorski groups introduced the lock washer, represented by the displaced edge of the structure, occurs at the interface between the first (A, blue) and the seventh (G, gray) helices. tetratricopeptide repeat (TPR) protein.[75,76] TPR, comprised b) Helical wheel of the sequence of peptide 7HSAP1(left) and schematic of 34 amino acids as a basic repeat unit, was originally iden- representation of the proposed model for self-assembly of lock-washer tified as a protein interaction module in the cell division structures derived from the seven-helix bundle of peptide 7HSAP1 into cycle proteins (Table 1).[75,76] The TPR unit could be found in helical nanotubes. c) The seven-helix bundle of GCN4-pAA that occupies unrelated functional proteins in a range of organisms from a structural homologous positions with respect to the isotopically labeled simple bacteria to human beings.[77,78] TPR mediates a variety sites in 7HSAP1*: carbonyl carbons (C’) of Ala9 (black), amide nitrogen of protein–protein interactions, thereby bringing together dif- of Leu13 (blue), and methyl carbons of Ala21 (gray). Reproduced with permission.[74] Copyright 2013, American Chemical Society. ferent proteins in complex biological machines.[78,79] The amphipathic nature of the TPR protein suggests that it might form a coiled-coil or helical bundle structure. However, short peptide having a sequence MEEVF.[73] This interaction is the three-TPR domain of phosphatase 5 as the earliest solved resistant to washing but can be disrupted under mild conditions TPR structure, showed that the structure was helical but the for purification purposes (Figure 11a).[80] A designed TPR module, folding presented a novel helical array.[77] Due to the consensus CTPR390+ binds the anticancer chaperone Hsp90 with high TPR motif being involved in protein–protein interactions, affinity and greater specificity than the endogenous cochaperones, redesign of the superhelix structure has been explored.[77] TPR enabling effective inhibition of Hsp90 functions that are essential domains can be used as a “switch” for either binding specificity to the folding of many oncogenic proteins (Figure 11b).[81] or to disrupt protein–protein interactions, resulting in a unique scaffold for protein engineering.[77] Regan and co-workers have designed recombinant con- 2.2. Multidomain Protein Engineered Materials sensus TPR proteins, manipulating the structure and stability in a rational way.[79] These TPR modules have been engineered Multidomain protein engineered materials are comprised into supramolecular arrays with practical applications including: of more than one domain within a polymeric chain.[82] Many 1) affinity purification capture reagent and 2) nanostructured func- of the single domains discussed in the previous section are tional films as novel anticancer agents.[79] affiTRAPs, designed employed to generate multidomain systems. Theoretically, each as TPR affinity proteins (TRAPs), are more cost efficient than domain or block of the protein material can self-assemble, pre- antibody-based affinity purification as it is easier to produce high serving each domain’s function, leading to an overall enhanced yields of these proteins. affiTRAP can specifically bind a target functionality of the multidomain system.

Figure 11. a) Schematic representation of all the components of the protein purification system. AffiTRAP is represented in blue and target protein in green. Upon washing, the cell lysate in pink can be easily washed out. By adding magnesium salt, the protein can be eluted. While upon addition of imidazole, affiTRAP is eluted. Reproduced with permission.[80] Copyright 2015, Portland Press Limited. b) Schematic representation of Hsp organizing protein indicating the two independent TPR domains, TPR1 and TPR2A, which interact with the C-terminal tails of Hsp70 and Hsp90 anticancer target. Red circle highlighted the TPR2A-Hsp90 interaction. Reproduced with permission.[81] Copyright 2008, American Chemical Society.

2.2.1. α-Helix (H)-Random Coil (RC)-α-Helix (H) Protein structures with heptad repeats (abcdefg)n. These helices can Polymers form a number of oligomers (dimers, tetramers, pentamers, hexamers, and octamers) having different topologies in the α-Helix-random coil-α-helix proteins (H-RC-H) represent a presence of random coil domain.[65] class of triblock proteins consisting of a random coil domain Tirrell and co-workers have constructed a triblock copolymer, flanked by either the same or different coiled-coil domain at AC10A (Figure 12a, Table 2), consisting of a central polyelec- [23] each end. A combination of α-helix coiled-coil and random trolyte random coil domain ((AG)3PEG)10 flanked by terminal coil can help tune the physical properties of the protein for a leucine zipper coiled-coil domains A, where a and d positions desired application. As previously discussed, right-handed of the coiled-coil are based on Jun oncogene product.[21,83] α-helices found in nature tend to self-assemble into coiled-coil The coiled-coil domain of AC10A, which self-assembles into a

Figure 12. a) Amino acid sequence and representation of secondary structure of a triblock polymer (AC10A) with a central polyelectrolyte domain, a [17] random coil (AG3PEG)x, flanked by terminal leucine zipper domains. Reproduced with permission. Copyright 1998, The American Association for the Advancement of Science (AAAS). b) The fast recovery of injectable protein hydrogel PC10P allows self-supporting structures to be produced. Reproduced with permission.[83] Copyright 2010, American Chemical Society.

Table 2. Sequences of multidomain proteins.

Type of protein Name of protein Sequence Reference

H- RC- H Protein AC10A MRGSHHHHHHGSDDDDKWA-SGDLENEVAQLEREVRSLEDEAAELEQKVSRLKNEIEDLKA-E [83] Polymers IGDHVAPRDTSYRDPMG-[AGAGAGPEG]10-ARMPTSGD- SGDLENEVAQLEREVRSLEDEAAELEQKVSRLK- NEIEDLKA-IGDHVAPRDTSMGGC *central polyelectrolyte block is Bold; Coiled-Coil domain is italic

Telechelic PC10P MRGSHHHHHHGSGDLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASGTSYRDPMG [17]

[AGAGAGPEG]10ARMPTSGSGDLAPQMLRE LQETNAA LQDVRELLRQQVKE ITFLKNT VMESDASGKLN

* P block is italic; C10 is Bold

Diblock and Triblock Block A: (VSSLESK)6 [84,85]

Copolymer Protein Block B: (AG)3PEG10

Block C:(VSSLESK)2-VSKLESK-KSKLESK-VSKLESK-VSSLESK

ABA H6VNADP[(VSSLESK)6]ASYRDPMG[(AG)2PEG]10ARMPTSADP[(VSSLESK)6] [85]

CBA H6VNADP[(VSSLESK)2-(VSKLESK)-(KSKLESK)-(VSKLESK)-(VSSLESK)]- ASYRDPMG[(AG)2PEG]10

ARMPTSADP[(VSSLESK)6]

ABC H6VNADP[(VSSLESK)6] ASYRDPMG[(AG)2PEG]10

ARMPTSADP[(VSSLESK)2-(VSKLESK)-(KSKLESK)-(VSKLESK)-(VSSLESK)]

CBC H6VNADP[(VSSLESK)2-(VSKLESK)-(KSKLESK)-(VSKLESK)-(VSSLESK)]- ASYRDPMG[(AG)2PEG]10

ARMPTSADP[(VSSLESK)2-(VSKLESK)-(KSKLESK)-(VSKLESK)-(VSSLESK)]

ZR−C10−ZR MGLEIRAAALRRRNTALRTRVAELRQRVQRLRNEVSQYETRYGPLKLRDWMG [63]

[(AG)3PEG]10ALMPVDLEIRAAALRRRNTALRTRVAELRQRVQRLRNEVSQYET RYGPLR

*C10 is Bold; ZR is italic H-RC-Protein H-S-H MRGS HHHHHH GSDDDDKA SGDLENE VAQLERE VRSLEDE AAELEQK VSRLKNE IEDLKAE [94] Polymers IGDHVAPRDTSYRDPMG AGAGAGPEG AGAGAGPEG AGAGAGPEG AGAGAGPEG AGAGAGPEGAGAGA- [91] GPEG AGAGAGPEG AGAGAGPEG AGAGAGPEG AGAGAGPEG ARMPT SGDLENE VAQLERE VRSLEDE AAELEQK VSRLKNE IEDLKAE IGDHVAPRDTSW *S is Bold; H is italic H-S-AdhD-H MRGSHHHHHHGSDDDDKWASGDLENEVAQL EREVRSLEDE AAELEQKVSR LKNEIEDLKA [90] EIGDHVAPRDTSYRDPMGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAG PEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGARMPHGMAKRVN AFNDLKRIGDDKVTAIGMGTWGIGGRETPDYSRDKESIEAIRYGLELGMNLIDTAEFYGAGHAEEIVGEAIKEF- EREDIFIVSKVWPTHFGYEEAKKAARASAKRLGTYIDLYLLHWPVD DFKKIEETLHALEDLVDEGVIRYIGVSNFN LELLQRSQEVMRKYEIVANQVKYSVKDRWP ETTGLLDYMKREGIALMAYTPLEKGTLARNECLAKIGEKYGKTAAQVALNYLIWEENVVA IPKASNKEHLKENFGAMGWRLSEEDREMARRCVGMPTSGDLENEVAQLEREVRSLEDEAA ELEQKVSRLKNEIEDLKAEIGDHVAPRDTSMGGC *S is Bold; H is italic; AdhD is underlined β-roll GSARDDVLIGDAGANVLNGLAGNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGV- [100] GYGHDTIYESGGGHDTIRINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQA- GIEKLVEAMAQYPD HS-Leuβ WASGDLENEVAQLEREVRSLEDEAAELEQKVSRLKNEIEDLKAEIGDHVAPRDTSYRDPMGAGAGAGPEGAGA- GAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAG- PEGAGAGAGPEGARMLGSARDDVLIGDAGANLLLGLAGNDVLSGGAGDDLLLGDEGSDLLSGDAGNDLLLG- GQGDDTYLFGVGYGHDLILESGGGHDTIRINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEII- HAANQAVDQAGIEKLVEAMAQYPD HS-DLeuβ WASGDLENEVAQLEREVRSLEDEAAELEQKVSRLKNEIEDLKAEIGDHVAPRDTSYRDPMGAGAGAGPEGAGA- GAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAG- PEGAGAGAGPEGARMLGSARDDLLLGDAGANLLLGLAGNDLLLGGAGDDLLLGDEGSDLLLGDAGNDLLLG- GQGDDLYLFGVGYGHDLILESGGGHDTIRINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEII- HAANQAVDQAGIEKLVEAMAQYPD *mutated β-roll is Bold

α-Helical- E [(VPGVG)2VPGFG(VPGVG)2]5VP [19] Elastin Protein [67] Polymers [102] C DLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASG

EC MRGSHHHHHHGSKPIAASA-E-LEGSELA(AT)6AACG-C-LQA(AT)6AVDLQPS

CE MRGSHHHHHHGSACELA(AT)6AACG-C-LQA(AT)6AVDKPIAASA-E-LEGSGTGAKLN

ECE MRGSHHHHHHGSKPIAASA-E-LEGSELA(AT)6AACG-C-LQA(AT)6AVDKPIAASA-E-LEGSGTGAKLN

Table 2. Continued.

Type of protein Name of protein Sequence Reference

CEC MRGSHHHHHHGSACELA(AT)6AAC-C-LQA(AT)6AVDKPIAASA-E-LEGSGT- C-LQALSI EPE MRCSSHHHHHHVDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVP- [103,104] GVGVPGVGVPGVGVPGEGVPGVGVPGVGELGSGLGSAPQMLRELQETNAALQDVRELLRQQVKEITFLKNT- VMESDASKLNTSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVP- GVGVPGEGVPGVGVPGVGVPGGLLECM T40A MRCSSHHHHHHVDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVP- GVGVPGVGVPGVGVPGEGVPGVGVPGVGELGSGLGSAPQMLRELQEANAALQDVRELLRQQVKEITFLKNT- VMESDASKLNTSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVP- GVGVPGEGVPGVGVPGVGVPGGLLECM *mutation is Bold Q54A MRCSSHHHHHHVDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVP- GVGVPGVGVPGVGVPGEGVPGVGVPGVGELGSGLGSAPQMLRELQETNAALQDVRELLRQAVKEITFLKNT- VMESDASKLNTSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVP- GVGVPGEGVPGVGVPGVGVPGGLLECM *mutation is Bold I58A MRCSSHHHHHHVDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVP- GVGVPGVGVPGVGVPGEGVPGVGVPGVGELGSGLGSAPQMLRELQETNAALQDVRELLRQQVKEATFLKNT- VMESDASKLNTSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVP- GVGVPGEGVPGVGVPGVGVPGGLLECM *mutation is Bold L37A MRCSSHHHHHHVDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPG VGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVG ELGSGLGSAPQMLREAQETNAAAQDVRELLRQQVKEITFLKNTVMESD ASKLNTSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEG VPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGGLLECM *mutation is Bold L37V MRCSSHHHHHHVDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPG VGVPGVGVPGEGVP- GVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVG ELGSGLGSAPQMLREVQETNAAAQDVRELL- RQQVKEITFLKNTVMESD ASKLNTSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEG VPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGGLLECM *mutation is Bold L37I MRCSSHHHHHHVDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPG VGVPGVGVPGEGVP- GVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVG ELGSGLGSAPQMLREIQETNAAAQDVRELL- RQQVKEITFLKNTVMESDA SKLNTSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVP GVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGGLLECM *mutation is Bold

Silk-Elastin Pro- SELP-47K MDPVVLQRRDWENPGVTQLVRLAAHPPFASDPMGAGSGAGS[(GVGVP)4GKGVP(GVGVP)3(GAGAGS)4]12 [108] tein Polymers (GVGVP)4GKGVP(GVGVP)2(GAGAGS)2GAGAMDPGRYQDLRSHHHHHH

SELP-415K MDPVVLQRRDWENPGVTQLVRLAAHPPFASDPMGAGSGAGS[(GVGVP)4GKGVP(GVGVP)11(GAGAGS)4]7(G

VGVP)11GKGVP(GVGVP)4(GAGAGS)2GAGAMDPGRYQDLRSHHHHHH

SELP-815K MDPVVLQRRDWENPGVTQLVRLAAHPPFASDPM[GAGS(GAGAGS)2(GVGVP)4GKGVP(GVGVP)11(GA

GAGS)5GAGA]6MDPGRYQDLRSHHHHHH E E Silk-Collagen CSSC and SCCS/ CS S C S: [(GA)3GE]24 [119] Protein Polymers and SECCSE C:LEKREAEAGPPGEPGNPGSPGNQGQPGNKGSPGNPGQPGNEGQPGQPGQNGQPGEPGSNGPQGSQGNP GKNGQPGSPGSQGSPGNQGSPGQPGNPGQPGEQGKPGNQGPAGG Two- Leucine Zipper Coiled LEIEAAALEQENTALETEVAELEQ EVQRLENIVSQYRTRYGPL-LEIRAAALRRRNTALRTRVAELRQRVQRL- [122]

Componentα- Coils ZE -ZR Pairs RNEVSQYETRYGPL

Helix-Elastin mCherry−ZE MGGSRSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDIL Polymers andα- SPQFMYGSKAYVKHPADIPDYLKLSFPEGFKW ERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRG Helix-Globular TNFPSDGPVMQKKTMGW EASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQ Protein LPGAYNVN IKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKSKLRGSGSLEIEAAALEQEN TALETEVAELEQEVQRLENIVSQYRTRYGPLRSHHHHHH

EGFP−ZE MGGSRSMASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVPWPTLVTTLCYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIF FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIM ADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQS ALSKDPNEKRDHMVLLEFVTAAGITHGMDELYNLRGSGSLEIEAAALEQENTA LETEVAELEQEVQRLENIVSQYRTRYGPLRSHHHHHH

*ZE and ZR is Bold

Table 2. Continued.

Type of protein Name of protein Sequence Reference MITCH CC43 WW Domain RLPAGWEQRMDVKGRPYFVDHVTKSTTWEDPRPE [124] Proline-Rich PPxY Peptide EYPPYPPPPYPSG C7 MGSSHHHHHHSSGLVPRGSSSGHIDDDDKVDGT[RLPAGWEQRMDVKGRPYFVDHVTKSTTWEDPRPE]

GTLDEL[AGAGAGPEG]2RGDSAGPEG[AGAGAGPEG]2ELLDGT([RLPAGWEQRMDVKGRPYFVDHVTKSTTWE

DPRPE]GTLDEL[AGAGAGPEG]2[RGDSAGPEG][AGAGAGPEG]2 ELLDGT)5[RLPAGWEQRMDVKGRPYFVDH VTKSTTWEDPRPE]GTLE

P9 MGSSHHHHHHSSGLVPRGSSSGHIDDDDKVDGT[EYPPYPPPPYPSG]GTLDEL[AGAGAGPEG]2ELLDGT([EY

PPYPPPPYPSG]GTLDEL[AGAGAGPEG]2ELLDGT)7[EYPPYPPPPYPSG]GTLE P1 EYPPYPPPPYPSGC [126] Reversible CaM-(8)-Zip MHHHHHHAADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTID- [128] Ca2+-Sensitive FPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDG-

Two-Component DGQVNYEEFVQMMTAK(AGAGAGPEG)8AGSGDLENEVAQLEREVRSLEDEAAELEQKVSRLKNEIED Hydrogel LKAE

PGD-(n)-PGD MHHHHHHAGHKKTDSEVQLEMITAWKKFVEEKKKK(AGAGAGPEG)nAGHKKTDSEVQLEMITAWKKFVEE KKKK

PGD-(n)-eNOS MHHHHHHAGHKKTDSEVQLEMITAWKKFVEEKKKK(AGAGAGPEG)nAGRKKTFKEVANAVKISASLMGAERLI tetramer, is responsible for pH controlled gelation and viscoe- applications.[83] Recently, they have developed a protein tri- lastic properties, while the random coil assists in the formation block copolymer in which the end coiled-coil block of AC10A of a highly swollen hydrogel.[21,83] The design of such revers- is replaced with the coiled-coil P derived from the N-terminal ible hydrogels is valuable for understanding and controlling helical domain of cartilage oligomeric matric protein (COMP) physical and biological properties such as strength, porosity that self-assembles into a pentameric structure, to yield PC10P and sensitivity that impact drug delivery and cell encapsulation (Figure 12b, Table 2).[17] This protein hydrogel demonstrates a

Figure 13. Representation of the four different morphologies of triblock polymer protein hydrogels (Polymers I, II, III, and IV refers to ABA, CBA, ABC, and CBC, respectively) characterized by SEM. Reproduced with permission.[85] Copyright 2005, American Chemical Society.

2.2.2. α-Helix (H)-Random Coil (RC) Fused to Globular Protein Domains

H-RC-globular protein copolymers represent a class of multidomain materials that contains an α-helix coiled-coil domain and a random coil domain along with a functionalized protein seg- ment.[88,89] A wide variety of such polymers self-assemble into protein hydrogels, resulting in multifunctional protein materials with enzymatic,[90,91] optically active,[88] analyte-binding,[92] or signaling abilities.[93] Banta and co-workers have generated a series of successful bifunctional fusion proteins by creating H-S diblocks[94] Figure 14. Coprecipitation of CaP and ZR−C10−ZR (left). SEM image of fused with other proteins including green fluorescent protein Single FS nanoparticle composed of nanoplate petals (right). Reproduced (GFP) and the tetrameric discosoma red fluorescent protein [63] with permission. Copyright 2016, American Chemical Society. (DSRED),[95,96] or enzymes like small laccase (SLAC),[89,97] a polyphenol oxidase from Streptomyces coelicolor, and AdhD,[90,98] transition from a linear elastic gel to sol at large strains, exhib- an aldo-keto reductase from Pyrococcus furiosus (Table 2).[89–91] iting shear thinning by three orders of magnitude.[17] These The H represents an α-helical coiled-coil derived from leucine unique properties allows the hydrogel to be injected through zipper domain (Table 2) while S is comprised of [AGAGA- [94] narrow gauge needles with moderate pressure, facilitating its GPEG]10. The bifunctional fusions combine self-assembly use in tissue engineering.[17] properties with the bioactivity profile of fluorescent pro- Kopeceˇk and colleagues have generated a series of stimuli- teins or enzymes. The resulting H-S-GFP-H, H-S-DSRED, responsive reversible protein block polymers, ABA, CBA, and H-S-GFP constructs are expressed and purified to form ABC, CBC (Table 2) that are capable of forming hydrogels hydrogels via physical crosslinking (Table 2, Figure 15a). All (Figure 13).[84,85] While block B is similar to the polyelectrolyte constructs assemble into elastic hydrogels having lower ero- [89] domain ((AG)3PEG)10 discussed above, block A has a sequence sion rates when compared to the parent H-S-H hydrogel. of (VSSLESK)6 exhibiting a transition temperature of 95 °C. More recently, they have developed a fusion protein containing Introducing lysine at key positions within block A results in a thermostable aldo-keto reductase (HS-AdhD-H) that self- block C (Table 2), lowering its transition temperature. The self- assembles to form a thermostable enzymatic hydrogel (Table 2, assembly of hydrogels are accompanied by intermolecular asso- Figure 15c).[90] The hydrogel retains the enzymatic activity of ciation of coiled-coil domain forming physically crosslinked the enzyme even at elevated temperatures.[90] Such bi-functional network.[85,86] Tailoring the amino acid sequence of the coiled- fusion proteins have tremendous potential for use in biofuel coil domain allows these triblock hydrogels to exhibit tem- cells, and as tissue engineering scaffolds.[88–91] perature- and pH-responsiveness.[85] The hydrogel assembly Banta and co-workers have also created calcium-dependent has been shown to be influenced by the overall length and hydrogels comprising of an α-helical leucine zipper domain (H) structure of the coiled-coil domain.[84] The proteins containing and a peptide motif derived from repeats-in-toxin (RTX) domain block A (ABA, CBA and ABC) are more stable at neutral con- of adenylate cyclase linked by a hydrophilic linker (S).[99] Intro- ditions rather than either acidic or basic pH, while the CBC duction of Ca2+ ions changes the peptide motif of RTX from protein polymer reveals the structure to be more stable from intrinsically disordered to a folded beta roll (β-roll) structure.[100] acidic to basic condition.[85] CBC forms reversible gel, which The β-roll structure is divided into two short parallel β-sheet is strongly elastic at room temperature and predominantly vis- faces separated by turns. Hydrophobic surface(s) resulting from cous at 55 °C. These engineered protein materials are useful mutating leucine residues on one (Leuβ) or both (DLeuβ) β-sheet in biomedical applications where stimuli responsiveness is faces, lead to self-assembly of HS-Leuβ and HS-DLeuβ hydrogels required.[87] (Table 2, Figure 15d).[99,100] The HS-Leuβ and HS-DLeuβ form Park et al. have designed hybrid flower-shaped (FS) nano- hydrogels in calcium rich environment, with HS-Leuβ requiring particles using an artificial recombinant protein ZR−C10−ZR additional crosslinking by the H domain; HS-DLeuβ creates a [63] (Table 2, Figure 14). ZR represents the helical domain derived hydrogel network at lower protein concentrations, without the [99,100] from a b-ZIP protein and C10 is a random coil block composed additional H domains. These scaffolds can be potentially of [(AG)3PEG]10. Addition of calcium chloride (CaCl2) to the tri- used for creating calcium dependent stimuli-responsive protein block copolymer in the presence of phosphate buffered saline based hydrogels and other biomaterials.[99] (PBS), results in hybrid FS nanoparticles with coprecipitation of calcium phosphate (CaP). These FS nanoparticles assemble into clusters that form porous higher order supraparticles at 2.2.3. α-Helix-Elastin Protein Polymers the air−water interface.[63] This colloidal system can be adapted by other similar systems for the production of complex supra- While H-RC-H protein polymers use α-helical coiled-coils structures.[63,87] Such colloidal systems can contribute greatly and random coil domains to form multifunctional hydrogels, to the study of biomineralization, biocatalysis, and biosensors coiled-coils have also been combined with ELPs. Montclare since the hybrid supraparticles provide high-affinity binding and co-workers have designed artificial protein block poly- sites for protein immobilization applications.[63] mers bearing two self-assembling domains (SADs), consisting

Figure 15. a) Representation of H-S-H with fluorescence proteins and their partial sequences (left). Schematic representation of H-S-GFP-H and H-S-DSRED physically crosslinked fluorescent protein hydrogels (right). Green and red ovals are fluorescent proteins GFP and DSRED respectively; bars represents H-domains. Reproduced with permission.[89] Copyright 2007, American Chemical Society. b) H-S-SLAC constructs with partial sequences. Reproduced with permission.[90] Copyright 2009, Elsevier Ltd. c) HS-AdhD-H constructs with partial sequences. d) Cartoon represents hydrogel formation of HS-Leuβ and HS-DLeuβ. Calcium ions are shown in red. The monomeric units consist an α-helical leucine zipper (yellow), a randomly coiled linker (purple), and either mutant Leuβ (light blue) or DLeuβ mutant (green). Reproduced with permission.[100] Copyright 2014, American Chemical Society. of the coiled-coil region of cartilage oligomeric matrix protein ECE demonstrating a viscoelastic behavior (Figure 16).[67] Of (COMPcc or C) and elastin domain (E).[13,66] Three different the three protein polymers, the CE diblock exhibited stronger constructs, EC, CE, and ECE have been assessed for their overall affinity to curcumin followed by ECE and EC, making CE as conformation, thermoresponsive behavior, and physicochemical an excellent drug delivery carrier capable of providing sustained properties (Table 2). Interestingly, the number of repeats and ori- release effects. Furthermore, EnC and CEn (n = 1–5) libraries entation of C and E domains impact the mechanical properties have been generated to study the effects of orientation of the with EC exhibiting elastic character, CE being viscous and two domains and the effect of the length of the E domain on

combine the high strength of silk domain with the elastic properties of the elastin domain.[50] For example, recombinant SELP47K has been fabricated into nanofi- brous scaffolds using the electrospinning technique (Table 2).[106] Three SELPs (SE8Y, S2E8Y, S4E8Y) with silk-to-elastin ratios of 1:8, 1:4, and 1:2 have been expressed in E. coli (Figure 17). The silk-to-elastin ratio governs the particle size and self-assembly of SELPs with the formation of micelle-like particles being predominantly affected by the amount of silk present.[14] By precisely tuning the silk-to-elastin ratio, different nanostruc- tures such as nanofibers, hydrogels, and nanoparticles have been generated with potential Figure 16. Cartoons of block polymers CE, EC, and ECE and how they self-assemble form net- [152] applications in biosensors, drug delivery, and worked soft gels. Reproduced with permission. Copyright 2012, American Chemical Society. tissue engineering.[105,107] In addition to the above listed applications, SELPs have been used for deliv- [101] [108–113] the physicochemical properties. The EnC library show elastic ering nucleic acids. The Ghandehari group has created behavior (above the transition temperature) that allow them to SELP47K and SELP415K (Table 2) having repeats of silk like [101] form soft gels, while the CEn library exhibit viscous behavior. domain (GAGAGS) with different numbers of elastin units Overall, the orientation of the C and E blocks influences the (GVGVP). In addition to GVGVP, modified elastin includes hydrogel formation and binding/release of small molecules.[19,67] lysine at the second position (GKGVP) (Figure 18a,b).[113] The Recently, a triblock protein, CEC has been created for forming molecular structure and composition of the SELPs greatly protein hydrogels (Table 2).[102] The addition of an extra C domain affect the physicochemical and mechanical properties including to CE improves the stability with CEC exhibiting enhanced elas- the time required for gelation, degree of swelling and shear ticity and increased small molecule binding ability.[102] modulus.[7,113,114] The gelation time and swelling ratio are The Tirrell group have produced the EPE protein (Table 2), influenced by the number of elastin repeats, temperature and which consists of a central domain derived from COMPcc (P) pH conditions. The higher the number of elastin repeats, the flanked by elastin-like domains (E). The tri-block consists of faster the gelation time and higher the swelling ratio; lower N- and C-terminal cysteine residues. Under denaturing condi- shear modulus indicates lower mechanical strength, enabling tions, EPE can covalently react with four-arm PEG vinyl sulfone the gel to spread around and deform.[114] These SELP hydrogels (PEG-4VS) via free thiol groups.[103] Upon removal of the dena- undergoes an irreversible sol-to-gel transition allowing DNA turant, the P domain associates to form physical crosslinks. In or viral particles to be loaded under aqueous conditions.[111] order to study the mechanical properties of the EPE hydrogels, SELPs (SELP47K, SELP415K and SELP815K) have also been six variants (T40A, Q54A, I58A, L37A, L37V, and L37I, Table 2) employed as matrices for controlled delivery of gene, targeting are created by mutating a single amino acid within the P domain head and neck tumors.[7,8,113–117] SELP815K (Table 2) hydrogel, at a and d positions based on the work of Montclare et.al.[66,104] designed to contain silk and elastin repeats at a ratio of 8:16, The variants Q54A and T40A exhibit a higher folded fraction has been encapsulated with reporter genes. Amongst the dif- of the P domain than I58A mutant, leading to stiffer gels. Fur- ferent variants, SELP815K shows the highest transfection effi- ther studies mutating leucine 37 which is known to decrease the ciency in in vivo tumor model.[108] These hydrogels serve as helicity of the P domain is explored. The variants L37A, L37V, a liquid chemoembolic, capable of codelivering (Figure 18c) and L37I demonstrate different relaxation behaviour with L37A chemotherapeutic drugs doxorubicin and sorafenib in an in revealing longer relaxation time compared to L37V and L37I. Dif- vitro system.[109] ferences of the sequence within the coiled-coil domain of each variant changes the energy necessary for mechanical deforma- tion at the macroscopic level. Hydrogels with complex relaxation dynamics have been also prepared by mixing block polymers with multiple relaxation times. Thus, programmed molecular genetics play an important role in controlling the dynamic relaxation behaivor in engineered coiled-coil protein based hydrogels.[104]

2.2.4. Silk-Elastin Protein Polymers Figure 17. Constructs of silk−elastin-like proteins that contain varying ratios of the silk-to-elastin blocks in each monomeric repeat. SE8Y (1:8), Silk-elastin like polypeptides (SELPs) consist of repeating S2E8Y (1:4), and S4E8Y (1:2). Reproduced with permission.[107] Copyright domains of silk and elastin.[50,105] These recombinant proteins 2011, American Chemical Society.

Figure 18. a) Schematic of SELP47K and SELP415K showing hydrogen bonds between silk-like blocks as sites of crosslinking between polymeric chains. b) Schematic of hydrogel structures formed by SELP47K, SELP415K, and SELP815K. Blue lines represent elastin blocks, red line represents silk blocks, and the black diamond represents covalent crosslinks between the silk blocks. Reproduce with permission.[114] Copyright 2010, Elsevier B.V. c) Schematic of liquid chemo-embolic process for localized release of doxorubicin and sorafenib. Reproduced with permission.[109] Copyright 2016, American Chemical Society.

2.2.5. Silk-Collagen Protein Polymers Both CSSC and SCCS are able to form nanotapes that self- assemble to form transparent gels when uncharged.[119] Inter- The Cohen Stuart group has generated self-assembling silk- estingly, the order of the blocks did not affect the properties collagen like block polymers, CSSC and SCCS (Table 2), of the gel except the gelation time. In a separate study, they where S is a pH–sensitive silk-like domain comprising of used the same block polymers, which they referred as CSESEC E E 24 repeats of (GA)3GE octapeptide and C is asparagine, serine and S CCS (Figure 19), to test the mechanical properties of and glutamine rich hydrophilic collagen-like domain.[118–120] the transparent gels. At low pH, both CSESEC and SECCSE can

Figure 19. TEM images (top) and AFM images (bottom) of CSSC (left) and SCCS (right) supramolecular structures. The white bar indicates 200 nm. It can be clearly seen from both the images that CSSC and SCCS form nanotape microstructures and self-assemble into fibrils. Reproduced with permission.[119] Copyright 2009, American Chemical Society. self-assemble to form similar nanotapes, ultimately forming These types of bioelectrocatalytic hydrogels can be used the gels.[118] Both the variants exhibit comparable elastic as biosensors and in tissue engineering and drug delivery modulus with SECCSE gelating much faster than CSESEC. applications.[91] These pH responsive gels have high potential for blood and tissue engineering applications.[119]

2.3. Two-Component Protein Engineered Materials

The above mentioned examples are focused on single component materials, where a single polymer chain can self-assemble to form particles, fibers, colloids and nanotapes.[57,63,69,119] While these assemblies possess interacting domains in the single polymer chain that leads to self-assembly, an alterna- tive approach is to combine two different protein chains that are able to interact with each other. In this section, we will discuss two component protein engineered materials resulting in more stable, controlled and functional systems.

2.3.1. Biocataytic Hydrogels from Two Components

Expanding on the H-RC-globuar protein motif, the Banta group has generated a bioelectrocatalytic hydrogel by mixing osmium bis-bipyridine ([Os(bpy)2Cl2]Cl) complexed H-S-H (referred to as metallopolypeptide, Table 2) with H-S-SLAC.[91] The [Os(bpy)2Cl2]Cl moiety is used as a redox-mediator while SLAC, an oxidoreductase, catalyzes the reduction of dioxygen to water. The helical domains within the metallopolypeptide Figure 20. Schematic showing crosslinking of the supramolecular hydrogel and H-S-SLAC can self-assemble into a tetramer coiled-coil [91] through: 1) formation of coiled coil, 2) dimerization of H-S-SLAC motif, structure forming a supramolecuar hydrogel (Figure 20). and 3) crosslinking of metallopolypeptide motif mediated by osmium bis- The mixed hydrogel demonstrates the ability to generate bipyridine. Reproduced with permission.[91] Copyright 2008, The National a catalytic current, thus enabling bioelectrocatalysis.[91] Academy of Sciences of the USA.

Figure 21. a) Representation of “rod-coil” and “globule-rod-coil” protein complexes and protein vehicles formed through the mixing of ZR that contains elastin-like protein (ELP, shown as blue coiled-coil structure) with its ZE counterpart that contains two different types of fluorescence protein (red globular protein: mCherry; green globular protein: EGFP). b) Confocal microscopy image of vesicles encapsulating fluorescein. Green fluorescence [122] indicates the fluorescein and red fluorescence indicates the mCherry-ZE. Reproduced with permission. Copyright 2014, American Chemical Society.

2.3.2. Two-Component α-Helix-Elastin Polymers “globule-rod−coil” protein complex when mixed with either [122] and α-Helix-Globular Protein mCherry−ZE or EGFP−ZE. These complexes self-assemble into thermally responsive coiled-coil protein vesicles capable The Champion group has used leucine zipper coiled-coils of encapsulating fluorescein and small molecules for drug [122] ZE-ZR pairs in which they fuse the ZR domain with an ELP delivery (Figure 21b). These biocompatible protein vesicles (Table 2) to yield ZR-ELP and the ZE domain with either can be used to incorporate enzymes or receptor ligands into mCherry or EGFP, resulting in mCherry−ZE or EGFP−ZE vesicle membranes for various practical applications and have (Table 2, Figure 21a).[121,122] As the ELP domain exhibits a potential as “carrier-free” protein delivery system capable of [123] phase transition behavior, the ZR-ELP forms a “rod−coil” and self-assembly within the extracellular matrix.

Figure 22. a) Schematic of MITCH. Protein polymers consisting repeats of the CC43 WW domain when mixed with proline-rich peptides, result in hydrogel formation. Reproduced with permission.[125] Copyright 2011, American Chemical Society. b) Schematic of SHIELD prepared by mixing together two components: C7 engineered protein and an eight-arm PEG-P1 with or without thermoresponsive PNIPAM. Reproduced with permission.[126] Copyright 2018, The Royal Society of Chemistry.

2.3.3. Mixing-Induced Two-Component Hydrogel (MITCH) 2.3.4. Reversible Ca2+-Sensitive Two-Component Hydrogel

Heilshorn and co-workers have developed mixing-induced two- Calmodulin, a calcium modulated protein, regulates many component hydrogels (MITCH) comprising of seven repeats of the calcium dependent processes found in eukaryotic cells.[127] Gal- CC43 WW domain (C7) and nine repeats of the proline-rich PPxY livan and colleagues have explored self-assembling materials peptide (P9) (Table 2, Figure 22a).[124] The C7 and P9 domains comprised of calmodulin and calmodulin binding domains, are selected since they possess multiple repeats with short amino exhibiting calcium dependent changes in microrheological acid sequences, exhibit specific interactions and do not interfere properties.[128] Three different triblock proteins are generated: with the cell signaling pathways.[124] MITCH demonstrates tun- one consisting of calmodulin and tetrameric leucine zipper able mechanical properties capable of encapsulating cells under domain connected via a hydrophilic protein sequence ((AG)3- [23,124,125] physiological conditions. In addition, C7 has been mixed PEG)8, referred as CaM-(8)-Zip and the other two triblock pro- with P1 that contains an eight-arm polyethylene glycol (PEG) teth- teins comprising of petunia glutamate decarboxylase (PGD) ered to poly(N-isopropylacrylamide) (PNIPAM) to create a shear- connected with either the human endothelial NO synthase thinning hydrogel for injectable encapsulation and long-term (eNOS) or another copy of PGD connected via the hydrophilic [126] [128] delivery (SHIELD) (Figure 22b). SHIELD minimizes destruc- linker ((AG)3PEG)n with n = 8,40 (Table 2). The CaM-(8)-Zip tion to the cell membrane during injection and improves viability is mixed with either of the PGD containing block proteins to of human induced pluripotent stem cell-derived endothelial cells form a two-component system leading to a calcium sensitive net- (iPSC-ECs) in vivo. The physically crosslinked hydrogels of the work (Figure 23a).[128] An increase in viscosity (about 5000-fold SHIELD family demonstrates potential in cell transplantation for increase) is observed when calcium is added to the mixture of treating peripheral arterial disease.[126] CaM-(8)-Zip and PGD-(8)-PGD, indicating the formation of a

Figure 23. a) Representation of structure and functions of each module. Modules can be combined together to produce different functionalized materials. b) Schematic of PGD-(8)-PGD and CaM-(8)-Zip formed network. Reproduced with permission.[128] Copyright 2006, American Chemical Society.

Adv. Healthcare Mater. 2019, 8, 1801374 1801374 (19 of 33) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advhealthmat.de hydrogel (Figure 23b).[128] These engineered proteins can be chemically linked hydrogel.[135] An increase in mechanical used to generate novel calcium sensitive materials useful in integrity is observed for these crosslinked ELPs, making them [135] tissue engineering and drug delivery applications. useful in cartilage tissue repair. In addition, ELP[KV6-112] and ELP[QV6-112] (Table 3) with X = lysine (K) or glutamine (Q) within the VPGXG repeating sequence, have been crosslinked 3. Chemically Crosslinked Protein Materials using an enzyme, transglutaminase (tTG) to form networked hydrogels (Figure 24).[136] These gels not only exhibit stabiliza- While the aforementioned examples focus on physically tion of the ELP matrix in situ, but can also promote cell viability crosslinked protein materials, in this section, we explore chemi- and cartilage matrix synthesis and accumulation.[136] These cally crosslinked protein-engineered materials, which prevent findings promote the use of ELP gels as injectable materials [129,130] [135,136] gel dissolution through covalent networks. Chemically and functional cartilage repair. Furthermore, ELP[KV7F] crosslinked protein-engineered materials have the ability to and ELP[KV2F] can be crosslinked using β-[tris(hydroxymethyl) form hydrophilic networks that absorb large amounts of solu- phosphino]propionic acid (THPP) to form hydrogels tion, yet remain insoluble and maintain their specific struc- (Figure 24).[137] The reaction takes place under mild aqueous ture.[23,131] Crosslinked protein hydrogels are classified as conditions. The resulting gels maintain cell viability, and their promising biomaterials[129] and have been of great interest in mechanical strength is comparable to the tissues found in car- a variety of biomedical[129,132,133] and pharmaceutical[131,134] tilage.[137] Overall, these crosslinked gels have a tremendous applications. potential in tissue engineering. Tirrell et al. have replaced X within (VPGXG)n of ELPs with the charged residue lysine, generating (VPGKG)n with 3.1. Chemically Crosslinking through Side Chains pH-responsive properties and crosslinkable sites (Table 3).[16] The lysine residues have been crosslinked using bifunctional Chilkoti and co-workers have explored several chemical and electrophiles.[16,138] Two different sequences of ELPs with enzymatic crosslinkers for ELPs, (Figure 24) to be used in its both lysine and isoleucine as the guest residues are generated applications as potential injectable materials and scaffolds for (Table 3). Lysine residues are introduced as crosslinking sites [27,135–137] tissue engineering. The lysines within ELP[KV6] or whereas isoleucine residue decreases the Tt to below ambient [138,139] ELP[KV16] (Table 3) are crosslinked with tris-succinimidyl ami- temperature for facile processing. Bis(sulfosuccinimidyl) [135] notriacetate (TSAT) to form a networked gel. The difference suberate (BS3) can react with ELPs via primary amine groups to in molecular weight, concentration, and lysine content signifi- generate protein films (Figure 25). By varying the ratio between cantly impacts the swelling and mechanical properties of the the crosslinker and the ELP, the tensile properties and moduli of the crosslinked elastin protein can be tuned. These protein films mimic the extracellular matrix and thus can be used as grafts for small blood vessels.[138] Focusing on ELPs, Conticello et al. have genetically engineered and expressed poly(Lys-25) based on the elastin-mimetic repeat sequence [(VPGVG)4(VPGKG)], which is crosslinked with an equivalent amount of bifunctional N-hydroxysuccinim- idyl (NHS) ester resulting in a well-defined microstructured hydrogel (Table 3).[140] A comparison of crosslinked protein hydrogel and uncrosslinked poly(Lys-25) reveals the hydrogel undergoes structural rear- rangement (macroscopic and microscopic) during phase transition (Figure 26a).[140] Its precisely defined structure permits a more rational evaluation of the effects of reaction conditions on its microstructure and materials properties.[18,21] In addition, they have generated a recombinant elastin- mimetic triblock copolymer, LysB10, comprised of hydrophobic sequences (IPAVG) that display plasticity separated by a central block that is both hydrophilic and elasto- Figure 24. Reaction scheme of ELPs with tris-succinimidyl aminotriacetate (TSAT), meric (VPGEG) with crosslinking sequence β-[tris(hydroxymethyl)phosphino]propionic acid (THPP) and enzymatic (tTG) crosslinker. (KAAK) inserted between the plastic-like Adapted with permission.[137] Copyright 2007, American Chemical Society. and elastic-like domain (Table 3, Figure 26b);

Table 3. Sequences of chemically crosslinking proteins.

Type of Protein Materials Name of protein Sequence Reference

Chemically Crosslinking ELP MSKGPG(VGVPGVGVPGGGVPGAGVPGVGVPGVGVPGVGVPGGGVPGAGVPGGGVPG)9 [27] Protein Hydrogel WPTer

ELP[KV6] ELP[KV16] VPGKG(VPGVG)6 [135]

VPGKG(VPGVG)16

ELP[KV6-112] ELP[QV6-112] MSKGPG[VGVPGKGVPGVGVPGVGVPGVGVPGVGVPGVGVPG]16WPTerTer [136]

MSKGPG[VGVPGQGVPGVGVPGVGVPGVGVPGVGVPGVGVPG]16WPTerTer

ELPs, where X = Lysine (VPGKG)n [138] Two ELPs X position replaced with Sequence 1: [138] both lysine and isoleucine M-MASMTGGQQMG-HHHHHHH-DDDDK(LD-GEEIQGHIPREDVYHLYP-

G((VPGIG)2VPGKG(VPGIG)2)4VP)3-KLE Sequence 2: M-MASMTGGQQMG-HHHHHHH-DDDDK(LD-GEEIQGHIPREVDYHLYP-

G((VPGIG)2VPGKG(VPGIG)2)4VP)3-KLE

Poly(Lys-25) [(VPGVG)4(VPGKG)] [140]

LysB10 [VPAVGKVPAVG(IPAVG)4][(IPAVG)5]33[IPAVGKAAKVPGAG][(VPGAG)2VPGEG(VPGAG)2]28 [141]

[VPAVG-KAAKVPGAGVPAVG(IPAVG)4][(IPAVG)5]33 [IPAVGKAAKA] *elastic-like central block is Bold; plastic-like end block is italic; crosslinking block is underlined

ELP-Based Protein MASMTGGQQMG-HHHHH-DDDDK-LQ[LDAS-bioactive [145,146]

site-SA((VPGIG)2VPGKG(VPGIG)2)3]4LE RGD TVYAVTGRGDSPASSAA [142]

u1 YAVTGGTARSASPASSA u2 YAVTGTSHRSASPASSA u3 YAVTGDRIRSASPASSA

ELP-RGD with Crosslinking Site MASMTGGQQMG-HHHHH-DDDDK-LQ[LDAS- (TVYAVTGRGDSPASSAA)-[(VPGIG)2VPG [143]

KG(VPGIG)2]3]4 LE Crosslinking site is bold

A. gambiae fruit fly gene AQTPSSQYGAP [38]

RZ10 (AQTPSSKYGAP -AQTPSSKYGAP)5

RLP12 (GGRPSDSFGAPGGGN)12 [148] [147]

SpyTag AHIVMVDAYKPTK [154] SpyCatcher AMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWIS- [152] DGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHID AAA MKGSSHHHHHHVDAHIVMVDAYKPTKLDGHGVGVPGVGVPGVGVPGEGVPGVGVP- GVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGELAHIVM- VDAYKPTKTSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVP- GVGVPGVGVPGVGVPGEG VPGVGVPGVG VPGGLLDAHIVMVDAYKPTKLEWKK BB MKGSSHHHHHHVDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDG- KELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVT- VNGKATKGDAHIDGPQGIWGQLDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVP- GVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGELYAVTGRGDSPASSAPIATS- VPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVP- GEGVPGVGVPGVGVPGGLLDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKF- SKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFT- VNEQGQVTVNGKATKGDAHIDGPQGIWGQLEWKK A-LIF-A MKGSSHHHHHHVDAHIVMVDAYKPTKLDGHGVGVPGVGVPGVGVPGVGVPGVGVP- GVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGELSPLPIT- PVNATCAIRHPCHGNLMNQIKNQLAQLNGSANALFISYYTAQGEPFPNNLDKLCGPNVTDF- PPFHANGTEKAKLVELYRMVAYLSASLTNITRDQKVLNPSAVSLHSKLNATIDVMRGLLSNVLCR LCNKYRVGHVDVPPVPDHSDKEVFRKKKLGCQLLGTYKQVISVVVQAFTSVPGVGVPGVGVP- GEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGGLLDAHIVMVDAYK- PTKLEWKK *SpyTag is Bold; SpyCatcher is italic: LIF is underlined

Table 3. Continued.

Type of Protein Materials Name of protein Sequence Reference GB1-Sc MRGSHHHHHHGSMDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEW- [153] TYDDATKTFTVTERSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGAT- MELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKAT- KGDAHI GB1-St MRGSHHHHHHGSMDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD- ATKTFTVTEAHIVMVDAYKPTK

(GB1-St)2 MRGSHHHHHHGSMDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD- ATKTFTVTERSAHIVMVDAYKPTKRSMDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDN GVDGEWTYDDATKTFTVTERSAHIVMVDAYKPTK

(GB1-Sc)3 MRGSHHHHHHGSMDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEW- TYDDATKTFTVTERSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGAT- MELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG- KATKGDAHIRSMDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDAT- KTFTVTERSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSS- GKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDA- HIRSMDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTERS- GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWIS- DGQVKDFYLYPGKYTFVETA APDGYEVATAITFTVNEQGQVTVNGKATKGDAHI

TNfn3-(GB1-Sc)3 MRGSHHHHHHGSRLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLT- EDENQYSIGNLKPDTEYEVSLISRRGDMSSNPAKETFTTRSMDTYKLILNGKTLKGETTTEAVDAA- TAEKVFKQYANDNGVDGEWTYDDATKTFTVTERSGAMVDTLSGLSSEQGQSGDMTIEED- SATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVA- TAITFTVNEQGQVTVNGKATKGDAHIRSMDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYAN- DNGVDGEWTYDDATKTFTVTERSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDG- KELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVT- VNGKATKGDAHIRSMDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD- ATKTFTVTERSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSS- GKTIST WISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI

(GB1-St)4 MRGSHHHHHHGSMDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD- ATKTFTVTERSAHIVMVDAYKPTKRSMDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYAN- DNGVDGEWTYDDATKTFTVTERSAHIVMVDAYKPTKRSMDTYKLILNGKTLKGETTTEAVDAA- TAEKVFKQYANDNGVDGEWTYDDATKTFTVTERSAHIVMVDAYKPTKRSMDTYKLILNGKTLK- GETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTERSAHIVMVDAYKPTK

*SpyTag is Bold; SpyCatcher is italic: GB1 and TNfn3 is underlined

ELP [TVYAVYGRGDSPASSA-((VPGIG)2VPGKG(VPGIG)2)3]4 [151]

enabling both physical and chemical crosslinking.[141] Vapor sequence (Table 3) for cell adhesion or u1, u2, and u3 (Table 3) phase crosslinking can be introduced by reacting the lysine to be cleaved by urokinase plasminogen activator (uPA), an residues with glutaraldehyde after LysB10 forms a protein film enzyme secreted by neurites.[142,143] The lysine within the pro- by physical crosslinking. Chemical crosslinking maintains the teins are chemically crosslinked with NHS esters to form hydro- material’s integrity upon loading, thus enhancing the capacity gels, which are capable of supporting cell adhesion and neural of LysB10 films.[141] This elastin-mimetic protein polymer with growth.[142] In another example, ELP-RGD with crosslinking plastic-like mechanical responses allows for its applications in sites (Table 3) is chemically crosslinked with THPC resulting vascular and nonvascular biomedical studies.[141] in hydrogels capable of encapsulating cochlear cultures.[143] Heilshorn and colleagues have developed ELP-based hydro- These protein hydrogels maintain the structural integrity of gels crosslinked with the chemical crosslinkers N-hydroxysuc- the cochlea compared to the nonencapsulated cochlea.[143] The cinimide (NHS) ester (Figure 27a) or tetrakis(hydroxymethyl) THPC crosslinked ELP-RGD hydrogel matrix have also been phosphonium chloride (THPC) (Figure 27b).[142–144] Both these used to maintain the NPC stemness.[144] These gels have been chemicals react with primary amines (lysine residues) to form prepared with varied stiffness and degradability. While hydrogel a crosslinked network. These hydrogels have been used for degradation is essential for NPC stemness, gel stiffness has no neural regeneration,[142] as dental and orthopaedic implants,[145] specific correlation in maintaining the stemness.[144] The lysine as well as for maintaining 3-D culture of murine cochlea[143] and bearing ELP-RGD with crosslinking sites is also conjugated neural progenitor cell (NPC) stemness.[144] The recombinant using a photoactive covalent crosslinker, NHS–diazirine (NHS– proteins (Table 3) designed for neural regeneration consists of diazirine, succinimidyl 4,40-azipentanoate) (Figure 27c).[145,146] lysine bearing ELP and bioactive sites of either RGD containing The development of a modular photoactive protein gel is useful

for fabricating biomaterial coatings, films, and scaffolds.[146] The ELP-based protein (hydrogel) with NHS–diazirine, has been spin or dip coated on Ti6Al4V, a commercially available dental and orthopaedic titanium-based implant material.[145] UV exposure of ELP gel containing the photocrosslinker, allows the gel to be chemically conjugated with Ti6Al4V, which can improve the speed of osseointegration of titanium implants by increasing the bone-implant contact (Figure 27d).[145] The Liu group has created the resilin mimic RZ10 (Table 3) derived from the fruit fly gene (Table 3).[38] By replacing tyrosine with phenylalanine and adding lysine as chemical cross-linking sites, RZ10 can be readily crosslinked with tris(hydroxymethyl) phosphine (THP) to form hydrogel.[38] Furthermore, they have attached different cell-binding domains, such as the RGD motif and vascular endothelial growth factor (VEGF), to exploit the cloning scheme containing bioactive domains. These gels exhibit a high compressive modulus, can maintain the viability Figure 25. Reaction scheme of ELP proteins chemically crosslinked with of mesenchymal stem cells (MSCs) and has potential use in bis (sulfosuccinimidyl) suberate (BS3). Adapted with permission.[138] cartilage tissue engineering (Figure 28a).[36,38,44] In a separate Copyright 2003, American Chemical Society. study, Kiick and co-workers have created RLP12 (Figure 28b,

Figure 26. a) Picture (top) and cryo-HRSEM (bottom) images of poly(Lys-25) hydrogel showing macroscopic and microscopic gel behavior, respectively. At low temperature (4 °C), the gel is in its expanded state (left). At high temperature (40 °C), the gel is in its collapsed state (right). Reproduced with permission.[140] Copyright 1999, American Chemical Society. b) Schematic of LysB10 showing crosslinkable primary amine sites, hydrophobic plastic-like endblock and hydrophilic elastic-like central block. Reproduced with permission.[141] Copyright 2008, Elsevier Ltd.

Figure 27. Chemical structures of a) N-hydroxysuccinimide (NHS) ester and b) tetrakis(hydroxymethyl)phosphonium chloride (THPC). c) Schematic of chemical crosslinking of lysine residues. Lysine residues in ELPs react with the NHS–diazirine crosslinker to form amide bonds, thereby synthesizing a diazirine-modified ELP (ELP–D). Upon exposure to ultraviolet light, the diazirine group forms a highly reactive carbene intermediate, which can insert into a neighboring ELP chain. d) Schematic of proposed ELP gel conjugation to titanium substrate upon exposure to UV light. Reproduced with permission.[146] Copyright 2012, The Royal Society of Chemistry.

Table 3), a modified exon 1 repeat motif, having similar muta- Staudinger ligation mediated gelation happens at a much tions as RZ10. The additional lysine residues can react with slower rate when N3-ELP is mixed with triarylphosphine func- [tris(hydroxymethyl)phosphino]propionic acid (THPP) to form tionalized ELP. While the azide groups are introduced in the a crosslinked hydrogel network that possess high elasticity with side chains of lysine residues (of ELP) via diazo transfer pro- great resilience, and can be used in tissue regeneration.[147,148] tocol, BCN and triarylphosphine ELPs are obtained by reacting Azide-alkyne cycloaddition or click chemistry represents a the lysine residues with activated esters of BCN and the triar- bioorthogonal reaction that enables specific reaction without ylphosphine reagent.[151] ELP hydrogels formed via SPAAC can cross reacting with common biological functional groups.[149] encapsulate human mesenchymal stem cells (hMSCs), human In the context of protein crosslinking via click chemistry, side umbilical vein endothelial cells (HUVECs), and murine neural chains bearing azides and alkynes are required.[150] Heilshorn progenitor cells (mNPCs) with improved cell viability.[151] and co-workers have created ELP hydrogels that crosslink Recently, several groups have employed SpyCatcher-SpyTag via strain-promoted azide-alkyne cycloaddition (SPAAC) or chemistry to generate protein hydrogels.[152,153] First introduced Staudinger ligation for the application of cell encapsulation by Howarth and co-workers, SpyCatcher-SpyTag chemistry is (Figure 29).[151] SPAAC mediated gelation happens instanta- comprised of two peptide tags (SpyCatcher and SpyTag, Table 3) neously when ELP with azide moieties (N3-ELP) are mixed that can rapidly form an isopeptide bond between the Lys-31 and with ELP with bicyclo[6.1.0]nonyne (BCN) groups (BCN-ELP) Asp-117 of SpyCatcher and SpyTag (Figure 30).[152,154] SpyCatcher (Table 3) to form hydrogel network.[151] On the contrary, and SpyTag are split from the second immunoglobulin-like

Figure 28. a) Scheme showing RZ10 resilin-based protein bearing crosslinking sites and bioactive domains. Compressive properties and cell-interactions were studied for tissue engineering applications. Reproduced with permission.[38] Copyright 2012, American Chemical Society. b) RLP12, a resilin-like protein can to form a hydrogel by crosslinking with [tris(hydroxymethyl)phosphino]propionic acid (THPP). The gel formation can take place under mild conditions and by varying the crosslinker ratio can generate RLP12 hydrogels and films. Reproduced with permission.[148] Copyright 2011, American Chemical Society. collagen adhesion domain (CnaB2) from the fibronectin binding tetramer ((GSt)4), and TNfn3-(GB1-SpyCatcher trimer) (TNfn3- [154] [153] protein (FbaB) of Streptococcus pyogenes. Tirrell and Arnold (GSc)3) (Table 3). The (GSc)3 and (GSt)4 result in soft chemi- have used elastin-like proteins to explore the potential of using cally crosslinked hydrogels.[153] They are able to suspend human SpyCatcher-SpyTag chemistry to create chemically crosslink lung fibroblasts (HFL1) in TNfn3-(GSc)3 and added (GSt)4 to hydrogels.[152] They designate SpyTag as A and SpyCatcher form soft crosslinked hydrogel that can encapsulate HFL1 as B. The AAA protein (Table 3) bearing three SpyTags at the within its 3D network.[153] Also, they investigated the cumula- head, center, and tail of the elastin-like protein and BB protein tive release of the (GSc)3/(GSt)4 hydrogel loaded with cyan fluo- (Table 3) bearing two SpyCatchers at the end of each N- and rescence protein (CFP) to explore the capability of the hydrogel C-terminal of the elastin-like protein with an integrin-binding as a drug carrier.[153] Chemical crosslinking protein into hydro- site (RGD) and a matrix metalloproteinase-1 (MMP-1) cleavage gels using the SpyCatcher-SpyTag chemistry have great poten- site are biosynthesized .[152] AAA can react with BB to form a tial applications in tissue engineering and developing scaffolds protein hydrogel through the Spy network covalent crosslinking. for biomedical usage and drug delivery.[152,153] When mouse 3T3 fibroblasts are suspended in BB and reacted Protein crosslinking can be achieved via enzyme catalysis with AAA, the AAA+BB hydrogel successfully encapsulates such as using sortase A.[155] Sortase family has the ability to fibroblasts showing no signs of cytotoxicity. Furthermore, they carry out site-specific transpeptidation reactions.[155,156] Liu and design the SpyTag protein, A-LIF-A (Table 3), with a chimeric Xiao employ Staphylococcus aureus sortase A (SrtA) as a mole leukemia inhibitory factor (LIF) variant that can be recognized cular stapler enabling the crosslinking of (S)-carbonyl reductase by mouse embryonic stem cells (mESCs); when A-LIF-A is dis- II (SCRII) from Candida parapsilosis into dimers and trimers solved with BB, a hydrogel is formed, resulting in a 3D cell (Figure 31, Table 3),[157] SCRII is a short-chain alcohol dehy- culture while maintaining the pluripotency of stem cells.[152] drogenase/reductase with higher efficiency in biotransforma- Li and co-workers have utilized the SpyCatcher-SpyTag chem- tion and increased thermostability upon crosslinking.[157] SrtA istry on tandem modular proteins to engineer them into hydro- is an attractive tool for protein engineering since it not only gels.[153] They select a small globular protein, GB1, and the third can mediate crosslinking between proteins but also can engi- fibronectin type III (FnIII) from the human extracellular matrix neer bacterial surfaces by ligating protein to surfaces of gram- protein tenascin (TNfn3) to construct the Spy network. They positive bacteria as well as covalently attach proteins to solid design GB1-SpyCatcher (GSc), GB1-SpyTag (GSt), GB1-SpyTag supports including PEG hydrogels and crosslinked polymer [156,158,159] dimer ((GSt)2), GB1-SpyCatcher trimer ((GSc)3), GB1-SpyTag beads.

Figure 29. Crosslinking chemistry of engineered ELP hydrogel via strain-promoted azide-alkyne cycloaddition (SPAAC) for the application of cell encapsulation. ELPs are functionalized with azides (N3-ELP) and strained cyclooctynes (BCN-ELP). Upon mixing, the functionalized ELPs crosslink via SPAAC to form hydrogels. Reproduced with permission.[151] Copyright 2016, Wiley-VCH.

4. Metal Templating Protein Materials structure, stability, self-assembly, chemical reactivity and stimuli- responsiveness.[162] Nanocomposites comprised of engineered proteins that can template inorganic metal ions, possess promising applications as efficient biosensors, contrast agents, delivery vehicles and 4.1. Gold Nanoparticles and Nanocomposites therapeutics.[71,160,161] In this section, different protein engineered materials will be discussed to illustrate how templa- Kiick and colleagues have generated a recombinant polypeptide tion of metal nanoparticles enhance/alter properties including 17H6 that assembles into irreversible β-sheet that allows it to

Figure 30. a) Cartoon of SpyCatcher-SpyTag split from CnaB2. b) Mechanism of rapid isopeptide bond reaction between the Lys-31 and Asp-117 of SpyCatcher and SpyTag. Reproduced with permission.[181] Copyright 2012, National Academy of Sciences.

5. Protein Materials Bearing Noncanonical Amino Acids

Incorporation of noncanonical amino acids (NCAAs) within the proteins offers unique chemical and biological functionalities, paving the way to novel materials with extraordinary properties and functions.[12,163] Although NCAA incorporation can be technically challenging, requiring arduous optimization efforts in increasing protein yields or incorporating multiple/different NCAA within the same protein, recent advances and successful approaches in NCAA incorporation have expanded the genetic code, resulting in biomaterials with extraordinary and unique capabilities.[164] NCAAs can be introduced into recombinant proteins through either residue-specific incorporation or site- specific incorporation.[164–166] Residue-specific incorporation globally replaces a particular canonical amino acid with its NCAA analogue, whereas site-specific incorporation targets a Figure 31. Schematic illustration showing the SrtA-mediated crosslinking specific residue within the protein sequence.[164,165] of SCRII into dimers and trimers. Reproduced with permission.[157] Copy- right 2017, Nature Publishing Group. 5.1. Fluorination form fibers at pH 2.3 and at a temperature of 80° C (Table 4). The polypeptide consists of repeats of alanine rich motifs with Although fluorine atoms are not found in any of the twenty positively charged histidine patches that complex with nega- amino acids, incorporation of fluorine analogues into the pro- tively charged gold nanoparticles (GNPs) (Figure 32). The elec- tein have been shown to enhance their stability with improve- trostatic interaction between 17H6 fibers and GNPs ≈( 2–3 nm ments in physical and mechanical properties.[167] With the help size) results in formation of 1D arrays (Figure 32). These nano- of modern protein engineering incorporation techniques, fluo- particle arrays have potential applications in constructing opto- rine can be readily introduced into a protein structure. Further- electronic devices.[68] more, fluorination can be used to study the folding mechanism Using the engineered coiled-coils C and Q, Hume et al. have of proteins and activity of biological molecules.[168,169] templated gold nanoparticles exploiting the N-terminal His- Montclare and co-workers have studied the impact of fluori- tag. The nanocomposites C-GNP and Q-GNP exhibit distinct nation on fiber assembly, supramolecular assemblies and [167,170] morphologies when compared to His-tag cleaved Cx and Qx mechanical properties. Using the α-helical coiled–coils C and are further studied for their electronic properties (Table 4, and Q mentioned previously, incorporation of trifluoroleucine Figure 33a).[71] The His-tagged C and Q nanocomposites form (TFL) is achieved via residue-specific incorporation (Table 5, large aggregates that adsorb onto the glassy carbon electrode Figure 34a). The resulting C-TFL and Q-TFL exhibits an increase surface, thereby decreasing the peak current. In contrast, in α-helicity, resulting in enhanced drug binding ability, the His-cleaved protein-GNPs remain soluble and exhibit an improved thermal stability and enhanced fiber assembly at increase in current.[71] These assemblies aggregate upon the pH 8.0 compared to their nonfluorinated variants.[170] Fluorina- addition of trifluoroethanol, thus providing a strategy that can tion of the α-helix-elastin protein polymer through the residue- tune the supramolecular assemblies of these nanocomposites. specific incorporation of p-fluorophenylalanine (pFF) results The α-helix–elastin diblock copolymers comprised of C and E in pFF-EC, pFF-CE, and pFF-ECE (Table 5, Figure 34b).[167] As domains (as mentioned in section 2.2) has been employed for tem- fluorination affects the properties and stability of the target pro- plation of GNPs.[160] The presence of hexahistidine tag at the N-ter- teins, it has been used to tune the mechanical properties of the minus of E1C and CE1 assists in crystallization of GNPs leading proteins. The fluorinated block polymers exhibit more elastic to the production of protein-GNP nanocomposites (P-GNPs) character with superior mechanical properties when compared [160] [167] (Table 4, Figure 33b). The CE1-His6–GNP and E1C-His6-GNP to their nonfluorinated variants. Incorporation of TFL and nanocomposites demonstrate improved delivery of curcumin with pFF improves overall stability, mechanical properties and allows an increased uptake by breast cancer MCF-7 cells.[160] self-assembly of proteins into higher ordered structures.[167,170]

Table 4. Sequences of metal templating proteins.

Type of Protein Materials Name of protein Sequence Reference

Gold Nanoparticles and 17H6 MGH10SSGHIHM (AAAQEAAAAQAAAQAEAAQAAQ)6AGGYGGMG [68] Nanocomposites

Cx APQMLRE LQETNAA LQDVREL LRQQVKE ITFLKNT SKL [71]

Qx VKE ITFLKNT APQMLRE LQETNAA LQDVREL LRQQSKL

into elastin-mimetic protein polymers for up to twenty-two repeats. E. coli MRA30, a release factor -1 (RF-1) attenuated strain, is used to support multisite suppression at selected amber codon positions, thereby introducing NCAA at multiple sites with the elastin sequence.[173] Bpa and AzF have been used to test how each NCAA can efficiently incorporate at designated locations. They have shown that the ability to recognize the substrate and charge the tRNA differs; the AzF incorporation into elastin mimetic protein polymer exhibits higher yield than Bpa.[173] Through this study, Figure 32. Schematic of 17H6 fibrils bearing positively charged patches that interact with negatively charged gold nanoparticles through elec- they have introduced a way to incorporate multiple photo- trostatic interactions. Reproduced with permission.[68] Copyright 2009, crosslinkable residues at desired positions for enhancing Wiley-VCH. protein functions. Tirrell et al. have developed a fusion protein, ZE-ELF com- 5.2. Photocrosslinking prising of 1) a surface anchor domain that consists of an ELP bearing p-azidophenylalanines (ELF) and has the ability to pro- The approach of photocrosslinking proteins has been com- vide strong adhesion to hydrophobic surfaces;[121,174] and 2) a monly employed to study protein–protein and protein and cell protein capture domain composed of an acidic leucine zipper [171,172] [174,175] interactions. Recently, there has been a growing interest motif ZE (glutamic acid-rich leucine zipper motif). in using photocrosslinking as a strategy to generate protein- A second polypeptide, ZR-target protein, comprised of a based materials. Compared to chemical crosslinking, photo- target protein fused to a basic motif ZR (arginine-rich leu- crosslinking can be performed under milder conditions to cine zipper motif) is employed (Table 5, Figure 36).[121] The [173] generate novel biomaterials. ZE-ELF, when photocrosslinked, covalently attaches to a solid Conticello and co-workers have introduced new chemical support, enabling the ZR-target protein (green fluorescent functionalities into elastin mimetic protein polymers by incor- protein (GFP) and glutathione-S-transferase (GST)) to be cap- [173] [121] porating NCAAs at the guest residue position (Table 5). tured through the pairing of the ZE and ZR. Compared to The NCAAs para-benzoyl-L-phenylalanine (Bpa) and para- traditional methods such as physical adsorption or covalent azidophenylalanine (AzF) (Figure 35) have been introduced conjugation through cysteine and lysine residues, this leucine

Figure 33. a) Representation of His tagged C and Q proteins (left), as compared to His tagged cleaved Cx and Qx proteins (right) upon GNP templation. TEM images shown for both nanocomposites demonstrated very distinct morphologies. Reproduced with permission.[148] Copyright 2015 American Chemical Society. b) Gold nanoparticle (GNP) templated-synthesis strategy and P-GNP sequences. Reproduced with permission.[160] Copyright 2016, OMICS international.

Table 5. Sequences of NCAA bearing proteins.

Type of Protein Materials Name of protein Sequence Reference

Coiled-Coil Protein C-TFL MRGSHHHHHHGSIEGR APQMTFLRE TFLQETNAA TFLQDVRETFL [170]

TFLRQQVKE ITETFLKNT SKL

Q-TFL MRGSHHHHHHGSIEGR VKE ITFTFLKNT APQMTFLRE TFLQETNAA

TFLQDVRETFL TFLRQQSKTFL

Diblock or Triblock Copolymer EpFF [(VPGVG)2VPGFG(VPGVG)2]5VP [167] Protein with Two SADs CpFF DLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASG

pFF-EC MRGSHHHHHHGSKPIAASA-EpFF-LEGSELA(AT)6AACG-CpFF-LQA(AT)6AVDLQPS

pFF-CE MRGSHHHHHHGSACELA(AT)6AACG-CpFF-LQA(AT)6AVDKPIAASA-EpFF-LEGSGT- GAKLN

pFF-ECE MRGSHHHHHHGSKPIAASA-EpFF-LEGSELA(AT)6AACG-CpFF-LQA(AT)6AVDKPI- AASA-EpFF-LEGSGTGAKLN

Elastin Mimic Protein with NCAA Elastin Repetitive Protein (Val-Pro-Gly-Val-Gly)2 (Val-Pro-Gly-(Xaa)-Gly)(Val-Pro-Gly-Val-Gly)2 [173] Polymers

Elastin with Globular Protein ZE ZR Linker LEI EAAALE RTRYG ALEVQ ENTALET EVAELEQ EVQRLEN IVSQY PL [121] LEI RAA RR RNTALRT RVAELRQ RVQRLRN SQYET RYGPL

G(GS)6G

ELF [(VPGVG)2-VPGFG(VPGVG)2]5VPGC

ZE-ELP LEI EAAALE RTRYG ALEVQ ENTALET EVAELEQ EVQRLEN IVSQY

PL-G(GS)6G-[(VPGVG)2-VPGFG(VPGVG)2]5VPGC *p-azidophenelalanine is bold

Figure 34. a) Schematic of residue-specific incorporation of TFL using leucine auxotrophE. coli cells to produce fluorinated protein that allow protein fiber formation. Reproduced with permission.[167] Copyright 2012, American Chemical Society. b) The schematic shows the sequence of pFF-ECE construct containing six histidine tag near the N terminal with E domain highlighted in black and C domain highlighted in red. Residue-specific incorporation of pFF happens at the site of phenylalanine (Phe). Reproduced with permission.[170] Copyright 2015, American Chemical Society.

zipper-elastin protein engineered material presents high speci- 6. Conclusions ficity and stability due to its heterodimeric leucine zipper association. Protein engineered biomaterials have many advantages compared to natural or synthetic materials. In this review, we provide an overview of the different techniques used for protein functionalization including crosslinking, metal templation, and incorporation of noncanonical amino acids. Protein materials are discussed from the perspectives of domain designs that can self-assemble or can be mixed together to form protein- networks. Since chemical crosslinking is known to enhance the mechanical properties of the gels,[130] we have discussed various examples that corroborate the use of crosslinkers for various protein-based gels. Furthermore, we have described the exam- Figure 35. Structures of the NCAAs with photocrosslinking properties. ples highlighting the assembly of gold nanoparticles-templated

Figure 36. Schematic process of surface functionalization and coiled coil mediated immobilization of recombinant proteins on substrates. The phenylalanine residues in the ELF domain are partially replaced by a photoreactive nonnatural amino acid, para-azidophenylalanine. This moiety can be used to generate covalent linkages to substrates upon UV irradiation. ZE is incorporated as the protein capture domain, and the basic portion ZR is fused to target proteins as an affinity tag. Reproduced with permission.[121] Copyright 2005, American Chemical Society. proteins and their applications in drug delivery. Lastly, we have Acknowledgements briefly discussed functionalization via NCAA incorporation. This work was supported by NSF-DMREF under Award Number DMR Overall, protein engineered functional materials are advan- 1728858, NSF-MRSEC Program under Award Number DMR 1420073, tageous because: 1) they are able to combine properties from NSF BMAT under Award Numbers DMR 1505214 and ARO W911NF-15- both naturally found materials and chemically synthesized 1-0304, the NYU Shiffrin-Myers Breast Cancer Discovery Fund, and the materials;[176] 2) they can better simulate and interface with NYU CTSA grant UL1 TR000038 from the National Center for Advancing cells and tissue, which may effectively minimize foreign Translational Sciences, National Institutes of Health. immune responses;[177] and 3) they can surpass the limita- tions of existing biotechnological natural/synthetic materials.[21] Single domain protein materials such as elastin, silk Conflict of Interest or coiled-coil proteins can result in hydrogels with tunable The authors declare no conflict of interest. properties including temperature-dependent phase transition, tensile strength and drug storage capabilities.[23,57,66] However, different applications may require the protein materials to be able to adapt and possess multiproperties. Multidomain pro- Keywords tein materials provide a solution by combining various func- biomaterials, coiled-coils, engineered proteins, functional materials, tionalities. While many of the described single and multid- recombinant protein omain protein materials can self-assemble into a physically crosslinked network forming reversible hydrogels, the strength Received: October 28, 2018 Revised: February 25, 2019 of such hydrogels may not be sufficient. Therefore strategies to Published online: April 2, 2019 develop stronger and stiffer crosslinked protein materials are crucial, demanding the use of chemical crosslinkers. Further- more, templation of engineered proteins with inorganic metal ions enable the construction of various biological assemblies [1] N. H. C. S. Silva, C. Vilela, I. M. Marrucho, C. S. R. Freire, C. P. Neto, and composites with diverse functions. These assemblies have A. J. D. Silvestre, J. Mater. Chem. B 2014, 2, 3715. [2] D. Kaplan, K. McGrath, Protein-Based Materials, Birkhäuser,Boston been typically used as biosensors, contrast agents and delivery [71,160,161] 1996, pp. xiii–xx. vehicles. NCAAs are also incorporated within the pro- [3] H. Sun, Q. Luo, C. Hou, J. Liu, Nano Today 2017, 14, 16. teins to pave the way to novel materials with extraordinary [4] W. Kim, E. L. Chaikof, Adv. Drug Delivery Rev. 2010, 62, 1468. [178] properties and functions. [5] J. L. Frandsen, H. Ghandehari, Chem. Soc. Rev. 2012, 41, 2696. The use of functionalized proteins holds great promise to [6] H. Ghandehari, A. Hatefi, Adv. Drug Delivery Rev. 2010, 62, 1403. generate novel biomaterials. Different strategies can be used [7] M. Haider, A. Hatefi, H. Ghandehari, J. Controlled Release 2005, to modify or decorate these materials with a range of func- 109, 108. tional groups. For example, protein-materials templated with [8] A. Hatefi, J. Cappello, H. Ghandehari, Pharm. Res. 2007, 24, 773. iron oxide particles are being used for biomedical imaging.[179] [9] X. Hu, P. Cebe, A. S. Weiss, F. Omenetto, D. L. Kaplan, materialstoday Other approaches include the use of lithography techniques 2012, 15, 208. [10] L. Yin, C. Yuvienco, J. K. Montclare, Biomaterials 2017, 134, 91. to pattern protein-based materials.[180] These strategies have [11] M. B. Dickerson, K. H. Sandhage, R. R. Naik, Chem. Rev. 2008, been used to create biomimetics that are endowed with proper- 108, 4935. ties that are similar, or better than originally found in nature. [12] C. C. Liu, P. G. Schultz, Annu. Rev. Biochem. 2010, 79, 413. Overall, the use of protein-based functionalized materials pro- [13] P. Baker, J. Haghpanah, J. K. Montclare, in Polymer Biocatalysis and vides an attractive approach for generating advanced materials Biomaterials II (Eds: H. N. Cheng, R. Gross), American Chemical and devices. Society, Washington, DC 2008, pp. 37–51.

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