Impact of aromatic residues on the intrinsic disorder and transitional behaviour of model IDPs

Abstract

Understanding the interplay between order and disorder in intrinsically disorder proteins (IDPs), and its impact on the properties and features of materials manufactured from them, is a major challenge in the design of protein-based synthetic polymers intended for advanced functions. In this paper an elastin-like diblock co-recombinamer amphiphile (Phe-ELR) based on a hydrophobic block containing five phenylalanine (Phe) residues proximal to the carboxyl function of a glutamic acid (Glu) residue upon folding, and with Glu as the guest residue in the hydrophilic part, was engineered and its assembly behaviour compared with another amphiphilic ELR used as control. Phe-ELR was tailored in order to clarify the impact of the presence of aromatic residues in the amino acid sequence, which even in early studies by Urry's group already demonstrated a certain out-of-trend behaviour compared with other apolar amino acids, especially non-aromatic ones, on ELR behaviour. The combination of several experimental techniques indicates strong molecular interactions associated with the Phe residue, thus resulting in limited reversible character of the temperature-induced transitions during sequential thermal cycles, a lower than expected transition enthalpy, and clear differences in its supramolecular assembly with respect to the control ELR. A distinctive pre-aggregated state for the Phe-ELR under any condition of pH and temperature is found. Eventually, this state gives rise to Phe-core micelles or a solid jelly-like material, depending on the concentration, pH and presence of salts. In conclusion, it appears that the presence of aromatic residues and their ability to promote strong inter- and intramolecular interactions at any temperature and pH causes a complete modification of the order-disorder interplay present in other, non-aromatic ELRs. These molecular events have a profound impact on the physical properties of the resulting polymer when compared with other ELRs. This work helps to shed light on the limits that govern intrinsic disorder in ELRs beyond its inverse temperature transition.

Keywords: Elastin; Intrinsically disordered proteins; Protein block co-polymers; Self-assembling materials.

Graphical abstract

Fig. 1

Plots of normalized turbidity at 350 ​nm as a function of temperature; three consecutive heating and cooling cycles were applied. Some insets are included to appreciate in detail the change in optical density. PBS refers to phosphate saline buffer at pH 7.4 and mQ refers to Milli-Q water.

Fig. 2

CD spectra of Phe and Ile ELRs at a concentration of 0.3 and 0.1 ​mg/ml, respectively, at different pH values (2.5, 7 and 7.4), in different solvents (Milli-Q water and PBS) and at different temperatures (5 ​°C: blue lines, and 50 ​°C: red lines).

Fig. 3

TOCSY of Phe-ELR at pH 10 and 5 ​°C.

Fig. 4

NOESY of Phe-ELR at pH 10 and 5 ​°C.

Fig. 5

Superimposed ¹H NMR spectra of Phe-ELR at pH 2.5 (black line) and pH 7 (red line) at 5 ​°C.

Fig. 6

DSC thermograms showing three subsequent cycles for 150 ​mg/ml aqueous solutions of Phe- and Ile-ELRs at a heating rate of 5 ​°C/min. The inserts show the hydrogels formed (if any) after the 3 cycles (scale bar: 0.3 ​cm).

Fig. 7

Cryo-transmission electron microscopy (cryo-TEM) images showing representative assembled structures (samples were subjected to three heating and cooling cycles up to 50 ​°C: see sub-section 2.7 for detailed sample preparation) for Phe-ELR in water at pH 2.5 (A), pH 4 (B), pH 7 (C), pH 7.4 in PBS (D); and for Ile-ELR in water at pH 2.5 (E), and pH 7 (F). Scale bar 200 ​nm (the insets have the same resolution as the main image, so the same scale bar is applied to them).