A minimal length rigid helical peptide motif allows rational design of modular surfactants

Abstract

Extensive work has been invested in the design of bio-inspired peptide emulsifiers. Yet, none of the formulated surfactants were based on the utilization of the robust conformation and self-assembly tendencies presented by the hydrophobins, which exhibited highest surface activity among all known proteins. Here we show that a minimalist design scheme could be employed to fabricate rigid helical peptides to mimic the rigid conformation and the helical amphipathic organization. These designer building blocks, containing natural non-coded α-aminoisobutyric acid (Aib), form superhelical assemblies as confirmed by crystallography and microscopy. The peptide sequence is amenable to structural modularity and provides the highest stable emulsions reported so far for peptide and protein emulsifiers. Moreover, we establish the ability of short peptides to perform the dual functions of emulsifiers and thickeners, a feature that typically requires synergistic effects of surfactants and polysaccharides. This work provides a different paradigm for the molecular engineering of bioemulsifiers.

Figure 1. Design scheme and single crystal X-ray analysis of SHR-FLLF.

(a) Sequence modification of SHR-FF to afford SHR-FLLF with higher helical propensity. Major residues involved in sequence modification are highlighted. (b) Helical wheel representation of designed SHR-FLLF heptad peptide showed the predicted relative position of the amino acids in the helix. The width of the line decreases from the C terminus to N terminus. The flexible terminal Phe residues at e,f can favourably form π-stacking with Phe of neighbouring helical modules. The leucine residues present at a,d positions may afford supramolecular zipper structure owing to favourable hydrophobic interactions. (c) Depiction of canonical helical conformation of the two asymmetric units of SHR-FLLF observed in crystal as cylindrical helices. (d) Torsion angles of the asymmetric units (coloured round symbols) superimposed over ideal Ramachandran plot. (e) Packing of adjacent head-to-tail helical columns of SHR-FLLF revealed perpendicular packing pattern and stabilized by hydrophobic and stacking interactions as shown by coloured amino acid side chains (only relevant amino acid side chains are displayed). The Phe and Leu residues in yellow represent residues that participate in ‘knob into hole' structures. The Phe residues in red represent π-stacking interactions.

Figure 2. Validation of self-assembly and structural rigidity of SHR-FLLF.

(a–c) Cryo-TEM micrograph of cylindrical micelles-like nanofibres formed at concentrations of (a) 5, (b) 10 and (c) 15 mg ml⁻¹ in aqueous solution. Scale bars, 50 nm. (d) Viscosity versus shear rate profile of different concentration of peptides confirmed apparent viscoelastic behaviour on increasing peptide concentrations (from bottom to top; colour code: 5 mg ml⁻¹, blue; 10 mg ml⁻¹, green; 15 mg ml⁻¹, red). (e) FTIR peaks of SHR-FLLF at neutral (pH 7.4) (top) and acidic pH (bottom). (f) A view of the fibril-like structure from molecular dynamics simulation. The fibril-like structure consisted of four layers. Each layer is presented in a different colour. Inset: a zoom of π–π interactions viewed between the blue layer and the red layer.

Figure 3. Characterization of SHR-FLLF stabilized emulsion.

(a) Photographic image of silicone oil (20%) and water (80%) emulsions before and after emulsifications. Silicone oil was stained with Sudan III dye. (b) Fluorescence micrograph demonstrates the formation of oil-in-water emulsion. Oil droplets fluoresce because of entrapped Nile red dye. Scale bar, 10 μm. (c) Apparent viscosity versus shear rate profile of emulsions prepared with 5, 10 and 15 mg ml⁻¹ of SHR-FLLF, respectively (from bottom to top; colour code: 5 mg ml⁻¹, blue; 10 mg ml⁻¹, green; 15 mg ml⁻¹, red) showed thickening or stabilizing proficiency of peptide emulsifiers.

Figure 4. Demonstration of structural modularity of conformationally constrained helical peptide emulsifiers.

(a) Structural modification of SHR-FLLF to afford SHR-FLELF and SHR-FLELF. Major residues involved in sequence modification are highlighted. (b) Truncated FTIR spectra of SHR-FLELF (top) and SHR-FLKLF (bottom). (c) Photographic images depicting the long-term stability of emulsions prepared with SHR-FLELF (7.5 mg ml⁻¹) and SHR-FLELF (7.5 mg ml⁻¹). (d) Profiles of apparent viscosity versus shear rate of emulsions prepared with 5, 7.5 and 10 mg ml⁻¹ of SHR-FLELF, respectively (from bottom to top; colour code: 5 mg ml⁻¹, blue; 7.5 mg ml⁻¹, green; 10 mg ml⁻¹, red) established general thickening or stabilizing proficiency of this class of peptide emulsifiers.