Genetically encoded lipid-polypeptide hybrid biomaterials that exhibit temperature-triggered hierarchical self-assembly

Abstract

Post-translational modification of proteins is a strategy widely used in biological systems. It expands the diversity of the proteome and allows for tailoring of both the function and localization of proteins within cells as well as the material properties of structural proteins and matrices. Despite their ubiquity in biology, with a few exceptions, the potential of post-translational modifications in biomaterials synthesis has remained largely untapped. As a proof of concept to demonstrate the feasibility of creating a genetically encoded biohybrid material through post-translational modification, we report here the generation of a family of three stimulus-responsive hybrid materials-fatty-acid-modified elastin-like polypeptides-using a one-pot recombinant expression and post-translational lipidation methodology. These hybrid biomaterials contain an amphiphilic domain, composed of a β-sheet-forming peptide that is post-translationally functionalized with a C₁₄ alkyl chain, fused to a thermally responsive elastin-like polypeptide. They exhibit temperature-triggered hierarchical self-assembly across multiple length scales with varied structure and material properties that can be controlled at the sequence level.

Figure 1.

Schematic of the structure and synthesis of FAMEs through post-translational modification of ELPs. a) FAMEs consist of three main components: a myristoyl group (zigzag chain) and a structure-directing peptide sequence (green arrow, B) — which together form a PA domain — and an ELP domain (shown in red). b) Molecular structure of M-B₁-ELP shown as an example. In addition to the three main components, a short, flexible linker is also incorporated into the FAMEs, to ensure that myristoylation of the B domain is not sterically hindered by ELP (see text). c) Schematic representation of the one-pot expression and post-translational lipidation by tandem expression of the NMT enzyme (Δ1–35) and B_1–3–ELP using pETDuet expression vector in which B is a peptide that is designed de novo to be recognized by the NMT enzyme as a substrate, and myristoylated.

Figure 2.

Temperature-triggered macroscale self-assembly of FAMEs. a-i, Temperature-programmed turbidimetry assays and images (c, f, i) obtained after heating above T_c and cooling below T_t for B₁-ELP (a), M-B₁-ELP (b, c), B₂-ELP (d), M-B₂-ELP (e, f), B₃-ELP (g), and M-B₃-ELP (h, i). Each turbidimetry assay was carried out at three concentrations: 100 μM (solid lines), 50 μM (dotted lines), 25 μM (dashed lines). The arrows denote the onset of LCST behavior (T_t) (a,b) and the onset of hysteretic behavior and morphogenesis of M-B₂-ELP (e) and M-B₃-ELP (h) into macroscopic structures above the critical temperature (T_c). The images in c, f, and i demonstrate that the self-assembled structures formed by M-B₂-ELP and M-B₃-ELP on heating are stable upon cooling (f,i), whereas that formed by M-B₁-ELP resolubilizes (c).

Figure 3.

Spectroscopic and dynamic light scattering (DLS) characterization of the effect of myristoylation on the structure and the self-assembly of the FAMEs. a) DLS of FAMEs (M-B_1–3–ELPs), unmodified B_1–3–ELP, and controls at T < T_t. The shift in the DLS autocorrelation functions of the FAME’s to longer timescales compared to that of controls confirms the nanoscale aggregation of FAMEs below their T_t. Error bars represent the mean ± standard error of the mean (s.e.m., shown as a shaded band around each line) calculated from 12 measurements. b) ATR-IR of lyophilized FAMEs, B_1–3–ELPs, control PAs (M-B_1–3), ELP, and M-ELP. The secondary structure of controls and FAMEs have significant similarities, and myristoylation does not result in major changes in the secondary structure. Control PAs (grey solid line in each panel) form β-sheets in the lyophilized powder, and the internal structure of each PA is dictated by its B sequence. c) Variable temperature ATR-IR of FAMEs and control M-ELP at T = 30 °C (T_c > T > T_t, dashed lines) and at T = 50 °C (T > T_c, solid lines). The conformation of the ELP domain does not change significantly in each FAME above its T_t; however, the PA-domain has a subtle effect on the conformation of the ELP. Arrows mark the position of characteristic ELP peaks at amide I and II bands. d) Static ThT fluorescence quantifies the propensity to form β-sheets at T = 20 °C (T < T_t). Error bars represent mean ± standard deviations calculated from three measurements. e) Dynamic ThT fluorescence assay, which show that the temperature-triggered phase transition of ELPs triggers the self-assembly of PA-domains, and in turn the FAMEs (arrows mark the final stage in the hierarchical self-assembly of M-B₂-ELP and M-B₃-ELP).

Figure 4.

Characterization of the morphology of the FAME aggregates and visualization of their temperature-triggered phase transition and self-assembly across different length scales and temperatures. a-c, Cryo-TEM of the FAMEs dissolved in PBS at 20 °C (T < T_t): M-B₁-ELP (a), M-B₂-ELP (b), and M-B₃-ELP (c), showing cylindrical micelle morphology with an average length increasing with the β-sheet-formation propensity of the PA-domain. d-f, SFM topography of the FAMEs, drop cast from solution at 30 °C (T_c > T > T_t): M-B₁-ELP (d), M-B₂-ELP (e), and M-B₃-ELP (f), showing that FAME nano-aggregates are rod-like polymeric micelles and fibers. g-l, M-B₁-ELP transitions into liquid coacervates at 30 °C (g) that remain stable up to 50 °C (j) but reach a larger equilibrium volume. M-B₂-ELP transitions into a network of fibers above T_t (h). At higher temperatures (T > T_c), these fibers form a stable interconnected network of fibers (k). Above T_t, M-B₃-ELP forms a “beads-on-a-string” morphology that is likely due to the arrested coalescence of the initial liquid coacervates (i). Above T_c, these droplets form fractal-like amorphous aggregates (l). The inset in k and l provide a higher magnification obtained using the super-resolution mode of the microscope.

Figure 5.

SEM morphological characterization of the macroscopic aggregates formed by heating M-B₂-ELP (a) and M-B₃-ELP (b) above their T_c and fixing the samples with glutaraldehyde and dehydration. The insets are at 10x magnification.

Figure 6.

The proposed three-step mechanism of FAME self-assembly. Stage 1 (T < T_t): below T_t, the nanostructure of the aggregates is determined by the fine balance between the attractive forces of the PA-core and the repulsive force of the hydrated ELP-corona. Stage 2 (T_c > T > T_t): the dehydrated ELP domain undergoes a LCST phase transition into a liquid-like coacervate and tends to form spherical droplets. The PA domain modulates the size of, and interactions between, the ELP coacervates. Stage 3 (T > T_c): the repulsion between the ELP coronas —further dehydrated— is lessened, in turn decreasing the core-core distances inside the coacervates and drives macroscale self-assembly. The cores likely dynamically rearrange and non-covalently cross-link to form macroscopic aggregates.