Dual Self-Assembled Nanostructures from Intrinsically Disordered Protein Polymers with LCST Behavior and Antimicrobial Peptides

Abstract

Antimicrobial peptides (AMPs) have attracted great interest as they constitute one of the most promising alternatives against drug-resistant infections. Their amphipathic nature not only provides them antimicrobial and immunomodulatory properties but also the ability to self-assemble into supramolecular nanostructures. Here, we propose their use as self-assembling domains to drive hierarchical organization of intrinsically disordered protein polymers (IDPPs). Using a modular approach, hybrid protein-engineered polymers were recombinantly produced, thus combining designer AMPs and a thermoresponsive IDPP, an elastin-like recombinamer (ELR). We exploited the ability of these AMPs and ELRs to self-assemble to develop supramolecular nanomaterials by way of a dual-assembly process. First, the AMPs trigger the formation of nanofibers; then, the thermoresponsiveness of the ELRs enables assembly into fibrillar aggregates. The interplay between the assembly of AMPs and ELRs provides an innovative molecular tool in the development of self-assembling nanosystems with potential use for biotechnological and biomedical applications.

Figure 1.

(a) Molecular scheme of the modular design of the hybrid protein polymers (AMP-ELRs). Individual blocks (AMP, spacer and ELR) are not to scale. Additional information regarding the molecular weights can be found in the Supporting Information. (b) Copperstained SDS-PAGE of the pure recombinant products: SI ELR and the hybrid AMP-ELRs (GL13K-SI and 1018-SI). (c) Scheme of the thermally driven self-assembly of the ELR control, SI, into spherical micelles.

Figure 2.

Thermal behavior of the hybrid AMP-ELRs (a,b) and the ELR (c) monitored by turbidimetry. (d) Images of the protein polymer solutions at 37 °C (25 μM in ultrapure water). Evolution of OD as a function of temperature demonstrated that all protein polymers exhibit a reversible phase transition. The presence of AMPs contributed to the phase transition and thermal hysteresis. Solid red lines represent heating cycles, and dashed blue lines represent cooling ones.

Figure 3.

Negatively stained TEM micrographs of the ELR/AMP-ELRs after incubation at 5 °C. Presence of the AMPs drove the formation of nanofibers after short incubation periods (10 min, 1 h) which evolved over time, thus indicating a dynamic behavior.

Figure 4.

DLS intensity distributions of the three protein-engineered polymers below [(a) 5 °C] and above [(b) 37 °C] the T_t.

Figure 5.

Negatively stained TEM micrographs of the ELR/AMP-ELRs after incubation at 37 °C. The presence of the AMP drives a second self-assembly, which triggers the formation of hierarchical structures. Fibrillar aggregates with globular or amorphous shapes are found when the GL13K or the 1018 peptide, respectively, are found within the hybrid polypeptide.

Figure 6.

Molecular structures and molecular weights (M_W) of the GL13K and 1018 peptides. Ionic residues are marked in red and large hydrophobic residues are marked in green.

Figure 7.

(a) Intensity size distributions of the nanostructures formed by the hybrid protein polymers after the incubation below (5 °C) and above (37 °C) T_t. (b) Cryo-TEM micrographs of the GL13K-SI and 1018-SI samples after initial incubation at 5 °C for 24 h, where the AMP triggered fibrillar assembly, and (c) after subsequent incubation at 37 °C, where thermally triggered coacervation of the ELR drove aggregate formation. (d) Schematic representation of the hierarchical self-assembly of the hybrid polymers (AMP-ELR) and magnification of the nanostructures formed at physiological temperatures after the incubation at 5 °C.