Genetically Fusing Order-Promoting and Thermoresponsive Building Blocks to Design Hybrid Biomaterials

Abstract

The unique biophysical and biochemical properties of intrinsically disordered proteins (IDPs) and their recombinant derivatives, intrinsically disordered protein polymers (IDPPs) offer opportunities for producing multistimuli-responsive materials; their sequence-encoded disorder and tendency for phase separation facilitate the development of multifunctional materials. This review highlights the strategies for enhancing the structural diversity of elastin-like polypeptides (ELPs) and resilin-like polypeptides (RLPs), and their self-assembled structures via genetic fusion to ordered motifs such as helical or beta sheet domains. In particular, this review describes approaches that harness the synergistic interplay between order-promoting and thermoresponsive building blocks to design hybrid biomaterials, resulting in well-structured, stimuli-responsive supramolecular materials ordered on the nanoscale.

Keywords: disordered proteins; peptides; phase transitions; self-assembly; supramolecular assembly.

Figure 1.

Ordered-disordered recombinant polypeptides. a. Design of recombinant polypeptides was based on three disordered ELP components and four polyalanine ordered domains at amino acid percentages up to 50%. b. The material formed by E1–H5-25% shows a reversible transition from an optically translucent liquid to an opaque solid-like structure during a heating and cooling cycle. c. Optical density measurements showing hysteresis and no change in thermal behavior of the materials upon repeating cycles of heating and cooling. d. 20-μm-thick three-dimensional reconstruction of E1—H5–25% using super resolution microscopy. Scale bars are 50 μm and 10 μm for the inset. e. Mesoscale network architecture showing interconnected globular structures. Scale bars are 10 μm (left) and 1 μm (right). Reprinted with permission.^[52,54] Copyright, 2018, Springer Nature and Copyright, 2019, John Wiley and Sons.

Figure 2.

Self-assembly of vesicles or nanosheets from self-assembling bolaamphiphile fusion-protein building blocks, globule-Z_E/Z_R—ELP—Z_R. a) When the mixture is not rotated end-over-end, the formation of protein vesicles is observed; inset is a representative TEM micrograph of vesicles. b) Self-assembly of protein nanosheets under aqueous conditions directly from modular components; inset is a representative fluorescent micrograph of nanosheets. Adapted and reprinted with permission,^[103] Copyright 2022, American Chemical Society.

Figure 3.

(A) Schematic illustration showing recombinant fusion protein building blocks for membrane construction of single-layered vesicles (SLVs) and double-layered vesicles (DLVs), along with the locations of encapsulated hydrophilic and hydrophobic cargo. (B and C) Fluorescent micrographs showing encapsulation of diverse fluorescent dyes with different water solubility (fluorescein sodium salt, coumarin, calcein, Cy5, rhodamine B (octadecyl ester perchlorate) (RhoB—OEP)) in (B) SLVs and (C) DLVs. All scale bars are 2 μm. (D) Graphical representation of the MWCO of SLVs (red) and DLVs (black) with respect to the temperature. Adapted and reprinted with permission,^[105] Copyright 2023, Royal Society of Chemistry.

Figure 4.

a) Thermoresponsive behavior of the protein solutions. AFM images of the nanostructures for b) R4C and c) R8C at different temperatures. Adapted and reprinted from Luo et al, MDPI, open access.^[109]

Figure 5.

(a–d) Peptides designed for homotetrameric, antiparallel coiled-coil bundle formation that form high-aspect ratio polymers upon covalent assembly. a. Left, Bundlemer-forming peptides a and b (BFP_a and BFPb), shown in single-letter amino acid code, have at their N termini (blue) either maleimide (Mal) or cysteine (C). The carboxyl terminus (red) of each peptide is unreactive. Each sequence forms homotetrameric bundlemers: grey, BFP_b; white, BFP_a. Right, the thiol—maleimide click reaction yields chains with two covalent linkages between neighboring bundlemers. b. TEM of rigid rods produced with a 1/1 ratio of peptides BFP_a and BFP_b. The sample is negatively stained with phosphotungstic acid (PTA). c. CryoTEM of rigid rods longer than 1 μm in aqueous solution. d. Negatively stained TEM of short rigid-rod chains produced using an asymmetric ratio (10/9: [BFP_b]/[BFP_a]) of reacting bundlemers. Adapted with permission,^[113] Copyright 2019, Springer Nature. (e–i) Cryo—TEM reveals that genetic fusion of RLPs to both BFP_a (e–f) and BFP_b (g–h) mediates physical responsive assembly of coiled coil bundlemers into flexible nanofibrils. Samples were made with a polypeptide concentration of 12.5 μM in 25 mM phosphate buffer, pH 7.0 and assembled for 24 h at room temperature. Representative micrographs of multiple experiments are presented. Scale bars=500 nm. i) Rendering of coarse grain model of RLP₂—BFP_a assembly with alignment of BFP bundles in the nanofibrils (RLP indicated in blue and BFP_a indicated in orange). Adapted with permission,^[55] Copyright 2023, John Wiley and Sons.

Figure 6.

Representation of SELP analogs and corresponding nanogels formed by self-assembly: b) 47K, c) 415K, and d) 815K. Corresponding peak intensities of 1,8-ANS in SELPs: d) 47K, e) 415K, f) 815K, and g) unsheared 815K at varying concentrations in PBS. The corresponding peak intensities are plotted versus the logarithm of the polymer concentration and fitted with two linear regressions to determine critical formation concentration. Two linear regions indicated the soluble SELP below critical formation concentration and aggregated SELP above critical formation concentration. SEM images of lyophilized SELP nanogels with 1,8-ANS: h) 47K, i) 415K, j) 815K, and k) unsheared 815K. Scale bar represents 500 nm. Adapted and reprinted with permission,^[168] Copyright 2018, John Wiley and Sons.

Figure 7.

a) Scheme of the assembly of recombinant SELPs incorporated with photo-responsive CarHC peptide. The recombinant protein (SELPs with CarHC) assembled into a molecular network through AdoB12 induced CarHC tetramerization in the dark and disassembled upon exposure to light. Color code; Black block- CarHC domain; Red-green blocks- SELPs peptide. b) the gel-sol transition of SELPs—CarHC hydrogel under light. Adapted and reprinted with permission,^[175] Copyright 2021, Elsevier.

Figure 8.

a) Graphical depictions of SELP—815K and SELP—415K structures, respectively. Release profile of b) SELP—415K, and c) SELP—815K at a low and high concentration. d) Viscosity profile of SAGE containing polymer solutions over a temperature range simulating a typical intravesical administration. e) Rheology trace of polymers over a 2 h period demonstrates the distinct difference between SELP—815K, SELP—415K, Poloxamer 407, and PLGA—PEG—PLGA gelation profiles. Adapted and reprinted with permission,^[183] Copyright 2019, Elsevier.

Figure 9.

A general scheme of RLP—SLP copolymer time-dependent assembly and formation of self-supporting gels. Reprinted with permission,^[36] Copyright 2017, American Chemical Society.

Figure 10.

a) Genetic constructs that encode recombinant R4S2, R4S4, and R4S8 with varying ratios of resilin-to-silk blocks. Stress-strain curves of the gels b) under compression to failure and c) under tension to failure. AFM images of the nanostructures assembled from fresh solutions d) R4S2 and e) R4S8 at different temperatures. Adapted and reprinted with permission,^[59] Copyright 2021, American Chemical Society.