Directed destabilization of lysozyme in protic ionic liquids reveals a compact, low energy, soluble, reversibly-unfolding (pre-fibril) state

TL;DR

This study explores how the stability of lysozyme in protic ionic liquids is influenced by proton transfer, revealing a reversible, low-energy amyloid state that can unfold and refold multiple times before fibrillization.

Contribution

It demonstrates the reversible formation of a low-energy amyloid state of lysozyme in ionic liquids, linked to proton transfer, expanding understanding of protein stability and folding pathways.

Findings

Lysozyme refolds 97% within a specific proton chemical shift range.

Exceeding this range induces a reversible amyloid form.

Amyloid form has very low energy and can unfold/refold multiple times.

Abstract

Recent demonstrations of extraordinary stabilization of proteins in mobile protic [1] and aprotic [2] ionic liquid solutions at ambient temperatures have raised hopes of new biopreservation and drug transportation technologies. Here we examine the relation of folded protein stability to the state of the transferred proton [1], as determined by the N-H proton chemical shift, d(N-H). We identify a range of d(N-H) in which the unfolded lysozyme refolds 97%. Exceeding the stability range in the acid direction leads to the sudden formation and stabilization of a small, soluble, amyloid form of lysozyme which has its own stability range and which can again unfold/refold many times before an irreversible process, fibrillization, occurs. The tightly bound amyloid form of the lysozyme molecule, identified by circular dichroism spectra and dynamic light scattering, must be of very low energy since the unfolding process absorbs almost three times the enthalpy of normal lysozyme unfolding. alpha-lactalbumin shows similar behavior.