Elastin-mimetic Polypeptides Research Articles

Bilayer Hydrogel Composed of Elastin-Mimetic Polypeptides as a Bio-Actuator with Bidirectional and Reversible Bending Behaviors.

Biologically derived hydrogels have attracted attention as promising polymers for use in biomedical applications because of their high biocompatibility, biodegradability, and low toxicity. Elastin-mimetic polypeptides (EMPs), which contain a repeated amino acid sequence derived from the hydrophobic domain of tropoelastin, exhibit reversible phase transition behavior, and thus, represent an interesting starting point for the development of biologically derived hydrogels. In this study, we succeeded in developing functional EMP-conjugated hydrogels that displayed temperature-responsive swelling/shrinking properties. The EMP-conjugated hydrogels were prepared through the polymerization of acrylated EMP with acrylamide. The EMP hydrogel swelled and shrank in response to temperature changes, and the swelling/shrinking capacity of the EMP hydrogels could be controlled by altering either the amount of EMP or the salt concentration in the buffer. The EMP hydrogels were able to select a uniform component of EMPs with a desired and specific repeat number of the EMP sequence, which could control the swelling/shrinking property of the EMP hydrogel. Moreover, we developed a smart hydrogel actuator based on EMP crosslinked hydrogels and non-crosslinked hydrogels that exhibited bidirectional curvature behavior in response to changes in temperature. These thermally responsive EMP hydrogels have potential use as bio-actuators for a number of biomedical applications.

An Activation-Specific Platelet Inhibitor That Can Be Turned On/Off by Medically Used Hypothermia

Therapeutic hypothermia is successfully used, for example, in cardiac surgery to protect organs from ischemia. Cardiosurgical procedures, especially in combination with extracorporeal circulation, and hypothermia itself are potentially prothrombotic. Despite the obvious need, the long half-life of antiplatelet drugs and thus the risk of postoperative bleedings have restricted their use in cardiac surgery. We describe here the design and testing of a unique recombinant hypothermia-controlled antiplatelet fusion protein with the aim of providing increased safety of hypothermia, as well as cardiac surgery. An elastin-mimetic polypeptide was fused to an activation-specific glycoprotein (GP) IIb/IIIa-blocking single-chain antibody. In silico modeling illustrated the sterical hindrance of a β-spiral conformation of elastin-mimetic polypeptide preventing the single-chain antibody from inhibiting GPIIb/IIIa at 37°C. Circular dichroism spectra demonstrated reverse temperature transition, and flow cytometry showed binding to and blocking of GPIIb/IIIa at hypothermic body temperature (≤32°C) but not at normal body temperature. In vivo thrombosis in mice was selectively inhibited at hypothermia but not at 37°C. This is the first description of a broadly applicable pharmacological strategy by which the activity of a potential drug can be controlled by temperature. In particular, this drug steerability may provide substantial benefits for antiplatelet therapy.

Self‐Assembly of Thermally Responsive Amphiphilic Diblock Copolypeptides into Spherical Micellar Nanoparticles

As structure–property relationships for protein self-assembly have been elucidated, advances in chemistry and structural biology have facilitated the development of biologically inspired polypeptides through chemical and biosynthetic schemes that have afforded novel protein-based films, fibers, micelles, and gels. In a number of instances, reversible protein self-assembly has been driven by welldefined conformational changes of peptide units induced in response to an external stimulus. Indeed, designed molecular assembly of stimuli-responsive peptides has emerged as a “bottom-up” approach for creating complex, but ordered, hierarchical structures from simple amino acid building blocks. As illustrated by the design of diand triblock polypeptides, microand nanoscale features can be tuned by control of the amino acid sequence, molecular weight, and secondary structure of the peptide. In particular, amphiphilic block copolypeptides can self-assemble into a variety of diverse structures, including rods, cylinders, spheres, and vesicles. Although diblock copolymers consisting of chemically and conformationally distinctive individual polypeptide blocks have been produced by chemical and biosynthetic schemes, to date, relatively few recombinant amphiphilic diblock polypeptides have been synthesized. Given the capacity to incorporate targeting ligands, cell membrane fusion sequences, receptor activating peptides, fluorescent or chelating groups, as well as the ability to tailor pharmacokinetics, biodistribution, and peptide stability, significant opportunities exist for micelles or vesicles produced from recombinant protein block copolymers. Elastin-mimetic polypeptides based on the pentameric repeat sequence (Val-Pro-Gly-Xaa-Gly) undergo thermal and pH-responsive self-assembly in aqueous solution. Spontaneous phase separation of the polypeptide coincides with a conformational rearrangement of local secondary structure above a unique transition temperature (Tt) determined by the chemical identity of the fourth amino acid (Xaa) in the pentapeptide repeat. Recent studies have demonstrated the potential of engineered materials derived from elastin in a broad range of biomedical and biotechnological applications and, in particular, drug delivery. 8] Characteristically, elastin-mimetic blocks that contain hydrophobic amino acids in the fourth amino acid position, such as tyrosine, display a conformational transition from random coil to repetitive type II b turns at temperatures well below 37 8C, whereas blocks that contain a charged amino acid in this position, such as glutamic acid, persist as a random coil throughout the physiologic temperature range. Thus, we postulated that amphiphilic diblock copolymers bearing glutamic acid and tyrosine residues in Nand C-terminal blocks, respectively, would promote micelle formation by temperature-induced self-assembly with a core–shell structure. Moreover, we speculated that at a sufficiently high density of glutamic acid units, charge repulsion would limit the association of the hydrophilic blocks andminimize micelle aggregation. Micelles stabilized by self-assembly alone are typically unstable in a complex environment containing naturally occurring amphiphiles, such as plasma proteins, glycolipids, and lipopeptides. Therefore, by positioning cysteine residues between blocks, we hypothesized that highmolecular-weight protein aggregation or uncontrolled micelle–micelle association would be avoided by nanoparticle stabilization through disulfide cross-linking. These studies represent the first report of thermally responsive and crosslink stabilized protein micelles produced through the tailored design of recombinant amphiphilic diblock copolymers. Two amphiphilic diblock polypeptides (ADP1 and ADP2) were synthesized and self-assembled into micellar structures with consecutive cysteine residues incorporated at the core– shell interface (Scheme 1). Expression of the diblock synthetic genes in E. coli expression strain, BL21(DE3), afforded recombinant protein polymers in high yield after immobilized-metal-affinity chromatography (IMAC) purification from the cell lysate. Mass spectrometry confirmed a correspondence between the observed and expected masses of the respective diblocks with consistent sequence composition by amino acid analysis. The presence of cysteine residues within the polypeptide chain was characterized by the use of a thiolreactive fluorescent dye (see the Supporting Information). Differential scanning calorimetry (DSC) demonstrated an endothermic transition at around 10 8C for both diblock copolymers, which conforms to the established relationship between the position of the transition temperature and the mole fraction of tyrosine in elastin-mimetic protein poly[*] Dr. W. Kim, Dr. E. L. Chaikof Emory University Departments of Biomedical Engineering and Surgery Georgia Institute of Technology, School of Chemical Engineering 101 Woodruff Circle, Rm 5105, Atlanta, GA 30322 (USA) Fax: (+1)404-727-3667 E-mail: echaiko@emory.edu

Supramolecular assembly and small molecule recognition by genetically engineered protein block polymers composed of two SADs

Genetically engineered protein block polymers are an important class of biomaterials that have gained significant attention in recent years due to their potential applications in biotechnology, electronics and medicine. The majority of the protein materials have been composed of at least a single self-assembling domain (SAD), enabling the formation of supramolecular structures. Recently, we developed block polymers consisting of two distinct SADs derived from an elastin-mimetic polypeptide (E) and the alpha-helical COMPcc (C). These protein polymers, synthesized as the block sequences--EC, CE, and ECE--were assessed for overall conformation and macroscopic thermoresponsive behavior. Here, we investigate the supramolecular assembly as well as the small molecule binding and release profile of these block polymers. Our results demonstrate that the protein polymers assemble into particles as well as fully or partially networked structures in a concentration dependent manner that is distinct from the individual E and C homopolymers and the E+C non-covalent mixture. In contrast to synthetic block polymers, the structured assembly, binding and release abilities are highly dependent on the composition and orientation of the blocks. These results reveal the promise for these block polymers for therapeutic delivery and biomedical scaffolds.

Chemical Conjugation of Linear and Cyclic RGD Moieties to a Recombinant Elastin‐Mimetic Polypeptide ‐ A Versatile Approach towards Bioactive Protein Hydrogels

An elastin-mimetic polypeptide, (EMM)(7), with the amino-acid sequence GRDPSS [VPGVG VPGKG VPGVG VPGVG VPGEG VPGIG](7) was used for chemical conjugation of various integrin ligands (RGD peptides) to prepare bioactive hydrogels. The chemical approach involved (1) chemical protection of lysine residues with Fmoc or Boc groups, (2) chemical ligation of a protected linear or cyclic RGD ligand, with or without a hexanoic-acid spacer to the glutamic acid residue, (3) deprotection of the lysine functionalities and the RGD moieties and (4) cross-linking to form a bioactive hydrogel. (1)H NMR spectroscopy was used to quantify the multiple steps in the reaction. The chemical protection was found to be between 65 and 93% for Fmoc and Boc, respectively. The ligands studied included linear RGD cell-binding [H-FGRGDS-OH (1-l-RGD), H-Ahx--FGRGDS-OH (2-Ahx-FGRGDS) and a cyclic -H(2)N-(CH(2))(6)COHN-cyclo(-RGDfK-) (H-Ahx-c(-RGDfK-)) peptide also with a hexanoic-acid spacer. Cell adhesion with mouse osteoblast cells was dependent on the ligand type, ligand density and the use of a spacer.

Efficient Synthesis of Protein‐Drug Conjugates Using a Functionalizable Recombinant Elastin‐Mimetic Polypeptide

This report describes the efficient conjugation of doxorubicin-glycine-phenylalanine-leucine-glycine (1a) and rhodamine-glycine-phenylalanine-leucine-glycine (1b) units to a monodisperse elastin-mimetic polypeptide (EMM)(7) bearing eight primary amine groups for chemical attachment. The synthetic approach is based on the solid-phase synthesis of 1a and 1b followed by chemical conjugation to the elastin-mimetic polypeptide in the presence of HOBt/PyBob as activating agents to form the polypeptide conjugates 2a and 2b. Conjugation efficiency was 61.2% (4.9 doxorubicin units per polypeptide chain) for 2a and 53.7% (4.3 rhodamine units per polypeptide chain) for 2b, demonstrating the feasibility of using these tailor-made, recombinant polypeptides as potential drug carriers for cancer therapy.

A Stereoelectronic Effect on Turn Formation Due to Proline Substitution in Elastin-Mimetic Polypeptides

Stereoelectronic effects have been identified as contributing factors to the conformational stability of collagen-mimetic peptide sequences. To assess the relevance of these factors within other protein structural contexts, three polypeptide sequences were prepared in which the sequences were derived from the canonical repeat unit (Val-Pro-Gly-Val-Gly) of the protein material elastin. These elastin-mimetic polypeptides, elastin-1, elastin-2, and elastin-3, incorporate (2S)-proline, (2S,4S)-4-fluoroproline, and (2S,4R)-4-fluoroproline, respectively, at the second position of the elastin repeat. Calorimetric and spectroscopic investigations of these three polypeptides indicate that the incorporation of the substituted proline residues had a dramatic effect upon the self-assembly of the corresponding elastin peptide. The presence of (2S,4R)-4-fluoroproline in elastin-3 lowered the temperature of the phase transition and increased the type II beta-turn population with respect to the parent polypeptide, while the presence of (2S,4S)-4-fluoroproline in elastin-2 had the opposite effect. These results suggest that stereoelectronic effects could either enhance or hinder the self-assembly of elastin-mimetic polypeptides, depending on the influence of the proline analogue on the energetics of the beta-turn conformation that develops within the pentapeptide structural repeats above the phase transition. Density functional theory (DFT) was employed to model three possible turn types (betaI-, betaII-, and inverse gamma-turns) derived from model peptide segments (MeCO-Xaa-Gly-NHMe) (Xaa = Pro, 4S-F-Pro, or 4R-F-Pro) corresponding to the turn-forming residues of the elastin repeat unit (Val-Pro-Gly-Val-Gly). The results of the these calculations suggested a similar outcome to the experimental data for the elastin-mimetic polypeptides, in that type II beta-turn structures were stabilized for peptide segments containing (2S,4R)-fluoroproline and destabilized for segments containing (2S,4S)-fluoroproline relative to the canonical proline residue.

Biosynthesis of an Elastin‐Mimetic Polypeptide with Two Different Chemical Functional Groups within the Repetitive Elastin Fragment

A new protein engineering strategy was utilized to synthesize an elastin-mimetic polypeptide. The primary structure represents an elastic motif composed of thirty amino acids with one lysine and one glutamic acid per repeat unit EMM = (VPGVG VPGKG VGPVG VPGVG VPGEG VPGIG). The gene was constructed using a Seamless Cloning method by generating three DNA cassettes which all encoded the EMM repeat unit, but with different flanking restriction recognition sites. The DNA cassettes were assembled to yield a gene that could be directly cloned into the multiple cloning site of pBluescript II SK+. The resulting gene (EMM)(7) with approximately 650 base pairs in length was further cloned into the expression vector pET-28b. Protein biosynthesis in E. coli strain BLR(DE3) resulted in the 21.5 kDa repeating polypeptide His(6)-(EMM)(7) yielding up to 50 mg . L(-1) of cell culture. Secondary structure analysis by far UV circular dichroism revealed a minimum at 197 nm and a shoulder at 218 nm indicative for a random coil with some type II beta-turn conformation content. Lower critical solution temperature (LCST) behavior strongly depends on salt and polypeptide concentration. Importantly, first cross-linking experiments indicate successful hydrogel formation with a surface structure reminiscent to natural elastin as visualized by SEM micrographs.

Structure of an elastin-mimetic polypeptide by solid-state NMR chemical shift analysis.

The conformation of an elastin-mimetic recombinant protein, [(VPGVG)4(VPGKG)]39, is investigated using solid-state NMR spectroscopy. The protein is extensively labeled with 13C and 15N, and two-dimensional 13C-13C and 15N-13C correlation experiments were carried out to resolve and assign the isotropic chemical shifts of the various sites. The Pro 15N, 13Calpha, and 13Cbeta isotropic shifts, and the Gly-3 Calpha isotropic and anisotropic chemical shifts support the predominance of type-II beta-turn structure at the Pro-Gly pair but reject a type-I beta-turn. The Val-1 preceding Pro adopts mostly beta-sheet torsion angles, while the Val-4 chemical shifts are intermediate between those of helix and sheet. The protein exhibits a significant conformational distribution, shown by the broad line widths of the 15N and 13C spectra. The average chemical shifts of the solid protein are similar to the values in solution, suggesting that the low-hydration polypeptide maintains the same conformation as in solution. The ability to measure these conformational restraints by solid-state NMR opens the possibility of determining the detailed structure of this class of fibrous proteins through torsion angles and distances.

Self-assembly of block copolymers derived from elastin-mimetic polypeptide sequences

Protein polymers derived from elastin-mimetic peptide sequences can be synthesized with near-absolute control of macromolecular architecture using genetic engineering techniques. Elastin-mimetic diblock and triblock copolymers have been prepared using this approach in which the individual elastin blocks display different phase behavior in aqueous solution. The selective collapse of the more hydrophobic blocks above the lower critical solution temperature was employed to drive the thermo-reversible self-assembly of elastin-mimetic diblock and triblock copolymer into protein-based nanoparticles and nano-textured hydrogels, respectively. These materials display considerable promise as biomaterials for applications in drug delivery and soft tissue augmentation.

Elastin-based biopolymers: chemical synthesis and structural characterization of linear and cross-linked poly(OrnGlyGlyOrnGly).

Poly(OrnGlyGlyOrnGly) was synthesized by classical procedures in solution. The monomeric sequence -OrnGlyGlyOrnGly- was chosen as a modification of -ValGlyGlyValGly-, typical of elastin, to impart primary amine functionality, susceptible to cross-linking with appropriate bifunctional reagents. Herein we focus on the cross-linking of poly(OrnGlyGlyOrnGly) with glutaraldehyde. The polymers, both linear and cross-linked, were characterized and investigated for their molecular and supramolecular properties. Circular dichroism studies performed on linear poly(OrnGlyGlyOrnGly) revealed a variety of conformations similar to elastin. At a supramolecular level, different kinds of aggregates were found such as the elastin-like twisted-rope pattern of filaments and fibrils, together with other specific morphologies, similar to those recently identified in some elastin-mimetic polypeptides.

Chemical synthesis of cross-linked poly(KGGVG), an elastin-like biopolymer.

Previous studies afforded on peptides and polypeptides containing repetitive sequences of elastin have largely demonstrated that their molecular and supramolecular properties are fully representative of those of tropoelastin, the soluble, linear precursor of elastin itself. In the attempt to synthesize cross-linked elastin-mimetic polypeptides, the repeating sequence VGGVG (V: valine; G: glycine), typical of elastin, was modified to incorporate lysine residues, yielding the polymer poly(KGGVG) (K: lysine). This imparts primary amine functionality susceptible to cross-linking reaction with appropriate bifunctional cross-linking reagents. We report herein the chemical synthesis and cross-linking of poly(KGGVG) with glutaraldehyde (GTA) and with disuccinimidyl glutarate (DSG). In both cases, the characterization of the polymers, both linear and cross-linked, has been carried out by CD spectroscopy and transmission electron microscopy measurements. The obtained results, although not conclusive, demonstrate that poly(KGGVG), both linear and cross-linked, may be considered very similar to tropoelastin and mature elastin, as concerns its molecular and supramolecular properties.

Thermo-Reversible Self-Assembly of Nanoparticles Derived from Elastin-Mimetic Polypeptides

Thermo-Reversible Self-Assembly of Nanoparticles Derived from Elastin-Mimetic Polypeptides

Rapid Assembly of Synthetic Genes Encoding Protein Polymers

A general method is described for the rapid assembly of synthetic genes encoding protein polymers based on the Seamless cloning technique. Genes encoding repeats of the elastin-mimetic polypeptides [(Val-Pro-Gly-Val-Gly)4(Val-Pro-Gly-Xaa-Gly)] (Xaa = Lys 1; Ile, 2) were constructed using this technique. The use of this method eliminates the dependence of the concatamerization on a limited pool of nonpalindromic restriction endonucleases and reduces the number of subcloning steps. A synthetic gene of approximately 3000 base pairs in length was isolated that encoded a protein polymer based on repeat sequence 1. An inducible expression of this gene in bacterial cultures of recombinant E. coli afforded a 90 kDa protein polymer of 1 in high yield (64 mg/L of bacterial culture). The protein was purified to homogeneity using immobilized metal affinity chromatography. The sequence of the protein polymer was confirmed by N-terminal amino acid sequence analysis and MALDI-TOF mass spectrometry of site-specific proteolytic cleavage fragments.