Foldamer Stability Coupled to Aggregation Propensity of Elongated Trp‐Cage Miniproteins

Abstract

Here we present folding‐associated aggregation propensity of three Trp‐cage foldamers: E0 (20 aa), E5 (25 aa) and E10 (30 aa), models of different sizes but comparable molecular properties. Electronic circular dichroism (ECD), vibrational circular dichroism (VCD) and FT‐IR spectroscopic measurements were used to monitor their concentration‐dependent, heat‐induced (5 °C → 65 °C) “α→β” fold transition. The ECD curves of E0 display an ensemble of highly dynamic structures. ECD of both E5 and E10 foldamers show the expected Trp‐cage fold, dominated by their α‐helical properties. No sign of β‐structures was revealed by ECD at any conditions (5 °C < T < 65 °C, 5 < pH < 7, c ≈ 30 μM) for any of these miniproteins. However, at higher concentration (c ≈ 1–30 mM) both VCD and FT‐IR spectral features of E5 as well as E10 resemble that of a β‐strand (ca. 1615 cm–1), accompanied with “free β‐edges”, or native β‐sheets (ca. 1635 cm–1). E5 at lower concentrations (c ≈ 1–3 mM), and E10 at higher concentration (c ≈ 30 mM) display the α→native‐β→β‐sheet folding transitions, monitored by the characteristic C=O vibrational normal mode frequency shift as follows: ca. 1650 cm–1 → ca. 1635 cm–1 → ca. 1615 cm–1, respectively. The latter folding path is irreversible. The shortest polypeptide E0 has an “unordered” fold, while E10 presents the most tightly packed Trp‐cage 3D‐structure. We have found that both high dynamicity and/or tight molecular core packing are different in nature, but common in efficacy in preventing the polypeptide backbone chain against self‐aggregation. However, E5 is intermediate in size and stability, and thus among these three polypeptides it is the quickest to aggregate. The present molecular triad, E0, E5 and E10, serves as a good example of larger globular proteins for which aggregation and amyloid fiber‐like nanoparticle formations are often associated with Alzheimer's, Creutzfeldt–Jakob, or prion diseases.

Chiroptical Studies on Supramolecular Chirality of Molecular Aggregates

“…A number of chiroptical methods such as electronic circular dichroism (ECD), vibrational circular dichroism (VCD), and Raman optical activity (ROA) have been applied to reveal the static and dynamic features of chiral molecules . Among the methods, the VCD spectrum is an extension into the infrared and near‐infrared regions of the circular dichroism spectrum . A main advantage of the method has been its utility in determining the absolute configuration of a chiral molecule in a solution.…”

“…In addition to these aspects, VCD spectroscopy was recently applied to molecular aggregates . The attempts were motivated by the recent finding that the VCD signals are enhanced remarkably when chiral molecules aggregate …”

Aromatic Cluster Sensor of Protein Folding: Near‐UV Electronic Circular Dichroism Bands Assigned to Fold Compactness

“…Far‐UV ECD spectroscopy (185–260 nm) requires only a ∼50–100 μ m solution of protein to quickly and straightforwardly determine the structural characteristics of the polypeptide backbone chain. A U‐type ECD curve is associated with a “random coil” or highly dynamic protein structure, also established for Trp‐cage miniproteins . Although, E0, the shortest in the series, shows U‐type ECD characteristics, weak n→π* (∼220 nm) embedded in a strong negative band (∼205 nm) followed by a small positive [ Θ ] MR value below 190 nm, it also reflects some residual folded fraction as concluded also by NMR (Figure ).…”

“…AU -type ECD curve is associated with a" random coil" or highly dynamic protein structure,a lso established for Trp-cage miniproteins. [6] Although, E0, the shortesti nt he series, shows U-type ECD characteristics, weak n!p*( 220 nm) embedded in as trong negative band (~205 nm) followed by as mall positive [V] MR value below 190 nm, it also reflects some residual folded fraction as concluded also by NMR (Figure 1). If aT rp-cage miniprotein is mostly folded (e.g.…”

Role of water in protein folding, oligomerization, amyloidosis and miniprotein

“…Because the TC5b and its derivatives are excellent models for F/U studies in vitro and in silico conditions, it is an important question to investigate the aggregation propensity of these molecules, becuase aggregation is associated with misfolded diseases. The aggregation propensity versus stability of a folded globular protein is in inverse relationship, and this suggests the idea to explore the behavior of Trp‐cage proteins of different stability, that is, of different tightness of their cores . For this purpose, the differently truncated derivatives of EX‐4 were prepared with some mutations of the residues.…”