Physicochemical Characterization of a Biomimetic, Elastin-Inspired Polypeptide with Enhanced Thermoresponsive Properties and Improved Cell Adhesion

Figures

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  Figure 1. Comparison of part of the exon 26 sequence of elastins from different species. (A) Porcine (XP_020941438.1), ovine (XP_042096308.1), bovine (AAA30505.1), feline (XP_019676153.1), canine (XP_048967017.1), murine (NP_031951.2), rat (NP_036854.1), and human (AAC98395.1) homologues are aligned. In gray are the residues that are the most conserved among these species and that represent the consensus sequence for this region. Boxed, the pentapeptidic motif is followed by a tetrapeptidic block, thus forming the nonapeptidic repeat that characterizes this region. (B) Sequence of UELP hydrophobic domain. Gray, residues corresponding to the consensus; black, residues that are found in the human sequence and were maintained; white, residues that correspond to the consensus and differ from those of the human sequence and that were maintained to enhance the regularity of the repeated sequence; italics, motifs that were repeated to obtain a 50 amino acid domain; boxed, the elastin pentapeptidic repeats.

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  Figure 2. Comparison of the structure of the polypeptides inspired by the elastin human homologue. (A) Schematic representation of the primary structure of the UELP and HELP recombinant proteins. Black, his-tag; gray, cross-linking domains; and white, hydrophobic elastin-like domains. (B) Prediction of the secondary structure of the two biopolymers obtained by I- TASSER simulation. Purple, coil; light blue, helix; and gray, β-strand.

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  Figure 3. CD spectroscopic analysis of the two elastin-inspired polypeptides UELP (A) and HELP (B) at a concentration of 0.1 mg/mL as a function of temperature: blue line: 15 °C; purple line: 20 °C; green line: 35 °C; orange line: 40 °C; and red line: 45 °C.

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  Figure 4. Model of the minimized secondary structure of UELP and HELP obtained by the I-TASSER – MTD simulation.

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  Figure 5. Turbidimetric analysis of the human elastin-derived biopolymers as a function of temperature. UELP (A) and HELP (B) were solved at 2 mg/mL in 10 mM Tris buffer (open symbols) and in Tris/NaCl (solid symbols). Cooling turbidity profiles (in blue) were analyzed in Tris/NaCl buffer.

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  Figure 6. DLS diameters (intensity-based calculated values) for UELP (black symbols) and HELP (red symbols) in 10 mM Tris (A, C, respectively) and in 10 mM Tris/NaCl buffer (B, D) at a concentration of 2 mg/mL as a function of temperature ranging from 10 to 60 °C at a scanning rate of 0.5 °C/min. The vertical dashed bars show the respective Tt values; the horizontal arrows show the predominant size distribution.

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  Figure 7. DLS diameter (volume-based calculated values) as a function of temperature. Calculated values of particle distribution in Tris (open symbols) and Tris/NaCl buffer (filled symbols) at a concentration of 2 mg/mL for UELP (A) and for HELP (B).

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  Figure 8. Overlay of 1H NMR spectra of UELP (A) and HELP (B) in D2O (5 mg/mL) at 10, 30, and 60 °C. Arrows indicate the resonance peaks of the following amino acid residues: leucine + valine, proline, alanine, and phenylalanine + histidine.

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  Figure 9. IyTx/I10 °Cy as a function of temperature for UELP (A) and HELP (B), as determined by 1H NMR in D2O (5 mg/mL) in the range between 10 and 60 °C.

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  Figure 10. Representative phase-contrast images of cell cultures on coated and uncoated tissue-culture polystyrene wells (TP). MG-63 and NIH3T3 cell lines of human and murine origin, respectively, were seeded on uncoated TP wells (panels A and D) and on TP wells coated with HELP (TP -H, panels B and E) and UELP (TP -U, panels C and F) and grown under standard conditions. Images were acquired 24 h after seeding. The bar is 100 μm.

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  Figure 11. Representative phase-contrast images of cell cultures on coated and uncoated nontissue culture polystyrene (NP). MG-63 and NIH3T3 cell lines of human and murine origin, respectively, were seeded on the uncoated NP wells (panels A and D) and on the NP wells coated with HELP (NP -H, panels B and E) and UELP (NP -U, panels C and F) and grown under standard conditions. Images were acquired 24 h after seeding. The bar is 100 μm.