Case studies

Elastin Motifs

Elastin-like polypeptides have been widely studied as potential injectable biomaterials and implantable scaffolds for regenerative medicine and drug delivery (Chilkoti et al. 2002; Rodriguez-Cabello et al. 2007). Potential applications include cartilage and invertebral disk repair (Betre et al. 2006, Betre et al. 2002), small diameter vascular grafts (Liu et al. 2004), and spinal cord repair (Straley and Heilshorn 2009). Elastin motifs are attractive components of materials for tissue engineering because of their biocompatibility in in vivo studies (Nettles et al. 2008; Rincon et al. 2006), high expression levels in recombinant systems (Chow et al. 2006), and inverse phase transition, which allows simple purification (Chow et al. 2006). At low temperatures, proteins containing elastin-like motifs are soluble in aqueous solvents; however, at higher temperatures the proteins form a polymer-rich coacervate phase. This temperature sensitivity can be modified by altering the primary amino-acid sequence of the engineered protein (Urry et al. 1991). Temperature sensitivity has been used to trigger in situ gelation and drug release from elastin-like gels. These biopolymers are being explored as drug delivery vehicles for the sustained release of pharmacological agents to articular joints (Betre et al. 2006) and dorsal root ganglion (Shamji et al. 2008), thereby reducing the side effects usually associated with systemic drug delivery.

The temperature sensitivity of elastin-like proteins has also been used to prepare scaffold-free cell sheets for implantation studies (Zhang et al. 2006). By nonspe-cifically adsorbing engineered elastin-like proteins with cell-binding domains onto standard cell culture substrates, human amniotic epithelial and mesenchymal cells were able to form cohesive cell monolayers. These cellular sheets were then incubated for a short time at 4°C; this causes the elastin-like protein to swell and releases the cells from the surface as a cohesive cell sheet that can be transferred to another culture surface or used for implantation.

To form mechanically robust monolithic structures, elastin-like proteins have been electrospun into fibers (Huang 2000) and covalently crosslinked using bifunctional chemical crosslinkers (Di Zio and Tirrell 2003) or enzymatic crosslinkers (McHale et al. 2005). Two recent crosslinking strategies for elastin-like proteins are of particular interest. First, by incorporating a photoactive amino acid, p-azido-phenylalanine, into the elastin-like sequence, Carrico et al. created a material that could be photo-crosslinked into various patterns using standard lithography techniques (Carrico et al. 2007). Second, Lim et al. have included lysine amino acids with elastin-like proteins to enable rapid crosslinking with hydroxymethylphosphines (HMP) at physiological conditions (Lim et al. 2008). This novel crosslinking chemistry allows for in situ gelation of elastin-like proteins for cell encapsulation. The mechanical properties, and hence the cellular microenvironment, can be tuned by altering the location and number of lysine reactive sites contained within the engineered proteins.

Engineered elastin-like proteins have also been suggested as useful materials for potential mechanical applications. Atomic force microscopy studies of elastin-like proteins have been used to characterize the force required to stretch a single macro-molecule (Flamia et al. 2004). This knowledge was exploited by Diehl et al. to create a mechanical bridge that tethered together multiple molecular motors (Diehl et al. 2006). In another example, through modification with additional chemical moieties, elastin-like proteins may be useful as mechanically active light sensors (Carriedo et al. 2000; Strzegowski et al. 1994). Taken together, these results suggest that elastin-like polymers make an easily tunable system that tolerates substantial modifications of the primary amino-acid sequence to control the resulting material properties.

Silk Motifs

Natural silk has outstanding mechanical properties; however, silks can be very difficult to harvest from natural sources. For example, the strongest silks tend to be dragline silk secreted by canabalistic spiders that are unable to breed in captivity. Therefore, great efforts have been made to engineer recombinant silk materials. Synthetic genes encoding spider dragline silk from Nephila clavipes were successfully constructed, cloned, and expressed by Prince et al. (Prince et al. 1995). Synthetic gene technology has been widely used to control silk protein size, allowing the study of sequence length and structure-function relationships (Wong Po Foo et al. 2006), and to create engineered proteins with novel compositions such as silkelastin block copolymers (Cappello et al. 1990) for a variety of applications including scaffolds for tissue engineering (Bini et al. 2006; Haider et al. 2008), gene delivery (Haider et al. 2005), and drug delivery (Cappello et al. 1998; Megeed et al. 2002). A multitude of silk-types from various species (for example, spiders N. clavipes, Nephila edulis, Argiope aurantia, Nephilengys cruentata, Euprosthenops australis and silkworms Bombyx mori and Samia cynthica ricini) have now been recombi-nantly expressed in a variety of host systems, the most common being the bacteria Escherichia coli (Fahnestock and Irwin 1997; Lewis et al. 1996) and yeast Pichia pastoris (Fahnestock and Bedzyk 1997).

Much of the current work with silk motifs is focused on creating materials with tunable mechanical and biophysical characteristics based on the wide diversity of silk motifs found in nature. For example, a chimeric silkworm silk that included sequences from the domestic silkworm B. mori and the wild silkworm S. c. ricini was found to have improved solubility and an a-helical conformation unlike the P-sheet secondary structures present in native silk proteins (Asakura et al. 2003). Generally, engineered silks require the use of strong solvents and careful processing to overcome the tendency of the hydrophobic amino-acid sequences to aggregate into large coacervates. However, in a surprising discovery, Johansson et al. have recently expressed repetitive silk motifs from E. australis together with a nonrepetitive silk sequence and a hydrophilic tag to enhance solubility. By simply cleaving the hydrophilic tag from the engineered silk in aqueous solvent, the spontaneous formation of meter-long silk fibers was achieved (Stark et al. 2007).

A second goal in the study of silk-mimetic materials is to create novel chimeras that combine superior silk-like mechanical properties with specific biofunctionali-ties. As an example, Wong Po Foo et al. used the complex mineralized composite systems found in diatoms as inspiration for the design of materials with remarkable morphological and nanostructural details (Wong Po Foo et al. 2006). These materials featured multimeric RGD-functionalized domains of the major ampullate spidroin 1 (MaSp1) of N. clavipes spider dragline silk fused to the silica precipitating R5 peptide derived from the silaffin protein of the diatom Cylindrotheca fusi-formis. Purified fusion proteins were either cast into films or electrospun into fibers, and silicification reactions were conducted under mild conditions (pH 5.5 and room temperature) to yield substrates decorated with silica nanoparticles. Human bone marrow-derived mesenchymal stem cells (hMSCs) were able to grow and differentiate on these engineered silk materials due to the added biofunctionality of the RGD cell-binding motif. This study was the first to combine a structural domain with a functional cell-binding domain and an inorganic binding domain.

Coiled-Coil Motifs

Based on the common characteristics of physical hydrogels where rigid and soft segments coexist, Petka et al. created a multidomain artificial self-assembling protein (Petka et al. 1998), which consists of leucine zipper motifs as the rigid association segments and flanking flexible, water-soluble polyelectrolyte domains as the soft segments necessary to promote swelling. Leucine zipper motifs form part of a sub-category of coiled-coil domains found widely in nature and play key roles in the dimerization and DNA-binding of transcriptional regulatory proteins (O'Shea et al. 1989). These coiled-coils comprise six heptad repeats and fold into amphiphilic a-helices, which multimerize through electrostatic interactions. Hydrophobic interactions between interspersed nonpolar side chains further drive their association into oligomeric clusters (O'Shea et al. 1991). When two leucine zipper domains are linked together by a random coil polyelectrolyte domain, these triblock copolymers self-assemble into a three-dimensional coiled-coil polymer network at near-neutral pH (Petka et al. 1998).

Gelation is reversible in response to environmental fluctuations in pH, temperature, and ionic strength that disrupt the self-assembly of the leucine zipper domains. While the reversibility of these coiled-coil interactions is a useful property to design materials with controllable sol-gel transitions, the sensitivity of these physical interactions to environmental changes often results in slightly unstable and weak hydrogels. Several strategies were subsequently developed to stabilize the assembly of these coiled-coil domains into engineered protein physical hydrogels including the addition of cysteine residues (Shen et al. 2005), and the incorporation of noncanonical fluorinated amino-acid residues (Tang and Tirrell 2001) within the leucine zipper domains. Shen et al. successfully stabilized these protein-engineered hydrogels through the addition of cysteine residues, which bind to each other through covalent disulfide bonds (Shen et al. 2005). An alternative strategy used by Tang et al. was the successful incorporation of non-canonical hexafluoroleucine into the recombinant coiled-coil proteins through synthesis within an engineered bacterial host with a modified leucyl-tRNA synthetase (Tang and Tirrell 2001). Molecular dynamics simulation and experimental spectroscopic studies have shown that the hexafluorinated side chains resulted in more stable coiled-coil domain interactions, thereby increasing the thermal stability of the coiled-coil structures (Tang and Tirrell 2001; Yoder and Kumar 2002). Furthermore, the addition of noncanonical amino acids into engineered proteins adds novel chemical functionalities and physical properties to the materials. As an example, Zhang et al. introduced photoactive moieties within the elastin-like protein domains fused to coiled-coil domains via a hydrophilic spacer sequence (Zhang et al. 2005). The photoactive characteristics of the elastin-like domain allowed for its immobilization to surfaces while the coiled-coil domain remained available as a tethering site for target proteins with leucine zipper motifs. By fusing leucine zipper motifs onto various target proteins such as growth factors or peptide pharmaceuticals, hierarchical self-assembly of protein scaffolds can be created for various applications.

Calmodulin Motifs

Calmodulin motifs represent one of the more recent additions to the list of peptide domains included in a variety of protein-engineered materials. Calmodulin

(CaM) is a 16.5 kDa protein that regulates many Ca2+-sensitive pathways including neuronal communication and muscle contraction (Zhang and Yuan 1998). CaM undergoes a conformational change upon binding of four Ca2+ ions that allows it to reversibly associate with CaM-binding domains present in over 100 different proteins. Topp et al. used CaM motifs together with other peptide modules to create stimuli-responsive biomaterials that reversibly self-assemble (Topp et al. 2006). These biomaterials were designed to be sensitive to specific environmental chemical cues including pH and ionic concentration. Several tri-block modular proteins were designed using CaM as the sensory motif, leucine zippers as the self-assembling motif, random-coil hydrophilic spacers to confer flexibility and solubility, and various CaM-binding domains as the actuator motif. Depending on their molecular architecture, the hydrogels respond with predictable changes in their physical characteristics and mechanical properties to environmental stimuli. This study highlights a key advantage of modular protein engineering techniques, where a "genetic toolbox" of the various motifs are synthesized and used to create numerous materials of exact composition and known Ca2+ sensitivity using a systematic combinatorial approach. The ability to quickly synthesize a family of materials with exact molecular-level alterations should be quite useful both in fundamental studies of structure-function relationships in macromolecular systems and in optimization studies of material properties for specific applications.

Hall et al. have also taken advantage of the distinct functionality of CaM as a biosensor to make a calcium-modulated plasmonic switch (Hall et al. 2008). In this novel construct, a CaM domain was flanked by an inactive N-terminal cutinase domain and an active C-terminal cutinase domain. The latter covalently binds to a phospho-nate functional group that decorates nanoparticles on a coated monolayer surface. This particular design allows CaM to be unidirectionally oriented and evenly spaced on the surface. The added inactive N-terminal cutinase domain provides an additional mass, which results in a more significant change in overall protein packing density. Using a high-resolution localized surface plasmon resonance spectrometer, the real-time changes in dynamics, orientation, and structure of CaM with changes in Ca2+ ion concentration can be studied over long periods of time without photo-bleaching due to the absence of a fluorescence label. Additionally, due to the precise tunability of protein engineering, the CaM domain and the inactive N-terminal cuti-nase domain can be substituted by any peptide drug or protein of interest, such as a growth factor, to immobilize the latter on a surface and to present active site-directed ligands (Hodneland et al. 2002; Murphy et al. 2004). This immobilization approach of any engineered protein provides a powerful technique to study dynamics and conformation changes of an unlabeled protein in real-time and to immobilize specific biological molecules at known densities on surfaces for fundamental cell biology, tissue engineering, and protein science studies.

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