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Proline periodicity modulates the self-assembly properties of elastin-like polypeptides.

ABSTRACT: Elastin is a self-assembling protein of the extracellular matrix that provides tissues with elastic extensibility and recoil. The monomeric precursor, tropoelastin, is highly hydrophobic yet remains substantially disordered and flexible in solution, due in large part to a high combined threshold of proline and glycine residues within hydrophobic sequences. In fact, proline-poor elastin-like sequences are known to form amyloid-like fibrils, rich in β-structure, from solution. On this basis, it is clear that hydrophobic elastin sequences are in general optimized to avoid an amyloid fate. However, a small number of hydrophobic domains near the C terminus of tropoelastin are substantially depleted of proline residues. Here we investigated the specific contribution of proline number and spacing to the structure and self-assembly propensities of elastin-like polypeptides. Increasing the spacing between proline residues significantly decreased the ability of polypeptides to reversibly self-associate. Real-time imaging of the assembly process revealed the presence of smaller colloidal droplets that displayed enhanced propensity to cluster into dense networks. Structural characterization showed that these aggregates were enriched in β-structure but unable to bind thioflavin-T. These data strongly support a model where proline-poor regions of the elastin monomer provide a unique contribution to assembly and suggest a role for localized β-sheet in mediating self-assembly interactions.

SUBMITTER: Muiznieks LD

PROVIDER: S-EPMC3000959 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The lower critical solution temperature (LCST) of the thermo-responsive engineered elastin-like polypeptide (ELP) biopolymer is being exploited for the thermal targeted delivery of doxorubicin (Dox) to solid tumors. We examine the impact of Dox labeling on the thermodynamic and hydrodynamic behavior of an ELP drug carrier and how Dox influences the liquid-liquid phase separation (LLPS). Turbidity, dynamic light scattering (DLS), and differential scanning calorimetry measurements show that ELP undergoes a cooperative liquid-liquid phase separation from a soluble to insoluble coacervated state that is enhanced by Dox labeling. Circular dichroism measurements show that below the LCST ELP consists of both random coils and temperature-dependent β-turn structures. Labeling with Dox further enhances β-turn formation. DLS measurements reveal a significant increase in the hydrodynamic radius of ELP below the LCST consistent with weak self-association. Dox-labeled SynB1-ELP1 (Dox-ELP) has a significant increase in the hydrodynamic radius by DLS measurements that is consistent with stable oligomers and, at high Dox-ELP concentrations, micelle structures. Enhanced association by Dox-ELP is confirmed by sedimentation velocity analytical ultracentrifugation measurements. Both ELP self-association and the ELP inverse phase transition are entropically driven with positive changes in enthalpy and entropy. We show by turbidity and DLS that the ELP phase transition is monophasic, whereas mixtures of ELP and Dox-ELP are biphasic, with Dox-labeled ELP phase changing first and unlabeled ELP partitioning into the coacervate as the temperature is raised. DLS reveals a complex growth in droplet sizes consistent with coalescence and fusion of liquid droplets. Differential scanning calorimetry measurements show a -11 kcal/mol change in enthalpy for Dox-ELP coacervation relative to the unlabeled ELP, consistent with droplet formation being stabilized by favorable enthalpic interactions. We propose that the ELP phase change is initiated by ELP self-association, enhanced by increased Dox-ELP oligomer and micelle formation and stabilized by favorable enthalpic interactions in the liquid droplets.

Project description:BackgroundSafe and effective hemostatic materials are important for reducing mortality resulting from excessive hemorrhage. In this work, new biomaterials with hemostatic effects were created by fusing the gene coding for RADA-16, a self-assembling peptide with the sequence RADARADARADARADA, to the 3'-end of the open reading frame (ORF) encoding elastin-like polypeptides through gene recombination.ResultsThe fusion proteins, termed 36R, 60R and 96R, were solubly over-expressed in Escherichia coli BL21 (DE3) based on genetic manipulation of the high-efficiency prokaryotic expression vector pET28a (+) and bacterial transformation. Western Blot analysis showed that the over-expressed proteins were the target fusion proteins. The target proteins 36R with 94.72% purity, 60R with 96.91% purity and 96R with 96.37% purity were prepared using an inverse phase transition cycle at 65 °C followed by His-tag affinity chromatography. The proliferation results of the mouse fibroblast cell line L929 and hippocampus neuron cell line HT22 indicated that the fusion proteins did not cause obvious cell toxicity. The lyophilized spongy film of the purified 36R, 60R and 96R could stop the hemorrhage of a 2?×?2 mm bleeding wound in the mouse liver after 27.21?±?1.92 s, 18.65?±?1.97 s and 15.85?±?1.21 s, respectively. The hemostasis time was 21.23?±?1.84 s for rat-tail collagen and 14.44?±?1.33 s for RADA-16 lyophilized on gauze. The hemostatic time of three treated groups were all significantly superior to that of the negative control without any hemostasis treatment, which spontaneously stopped bleeding after 37.64?±?1.34 s. Statistical analysis showed that the spongy film with purified 96R exhibited an exciting hemostatic effect that was superior to rat-tail collagen and close to that of RADA-16 lyophilized on gauze.ConclusionsThese results revealed that the fusion proteins achieved by gene recombination technology could serve as a promising hemostatic material.

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Proline periodicity modulates the self-assembly properties of elastin-like polypeptides.

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