Insights into Conformational Ensembles of Compositionally Identical Disordered Peptidomimetics

While the conformational ensembles of disordered peptides and peptidomimetics are complex and challenging to characterize, they are a critical component in the paradigm connecting macromolecule sequence, structure, and function. In molecules that do not adopt a single predominant conformation, the conformational ensemble contains rich structural information that, if accessible, can provide fundamental understanding related to desirable functions such as cell penetration of a therapeutic or the generation of tunable enzyme-mimetic architecture. To address the fundamental challenge of describing broad conformational ensembles, we developed a model system of peptidomimetics comprised of polar glycine and hydrophobic N-butylglycine to characterize using a suite of analytical techniques, including replica exchange molecular dynamics atomistic simulations and liquid chromatography coupled to ion mobility spectrometry, which allowed us to distinguish the conformations of compositionally identical model sequences. However, differences between these model sequences were more challenging to resolve with characterization tools developed for intrinsically disordered proteins and polymers. These tools include double electron-electron resonance (DEER) spectroscopy and diffusion ordered spectroscopy (DOSY) NMR. Finally, we introduce a facile colorimetric assay that employs immobilized sequences leveraging a solvatochromic probe, Reichardt’s dye, to visually reveal conformational trends consistent with the experimental and computational analysis, thus providing a rapid and complementary method to characterize macromolecular disorder and unravel the complexity of conformational ensembles, either as an isolated or multiplexed technique.