Slow but complete, two state unfolding/refolding of lysozyme in "tuned" ~6M guanidinium carboxylate solutions

TL;DR

This study demonstrates that lysozyme can undergo slow but complete refolding in high-concentration guanidinium carboxylate solutions, with refolding efficiency influenced by solution composition and pKa tuning.

Contribution

It reveals conditions for pseudo two-state refolding of lysozyme in near-6M guanidinium solutions, highlighting the role of solution pKa tuning in folding kinetics.

Findings

Complete refolding achieved at 25°C in specific solutions

Refolding fraction depends linearly on log(waiting time)

Reducing Gdm+ concentration speeds up folding

Abstract

We using differential scanning calorimetry of the unfolding process to identify conditions in which the pseudo two-state refolding of thermally denatured lysozyme can be observed to occur on time scales of hours, in solutions that approach 6M in guanidinium cation, Gdm+. Remarkably, the fraction of lysozyme re-folded at 25 C reaches, and remains at 1.0. The refolded fraction is linear in log(waiting time), where the waiting time is the time at ambient temperature after an initial thermal denaturing (details in text) and immediate rapid cool to ambient. The favorable refolding conditions are achieved by tuning the solution anion composition to be comparable in pKa to, but somewhat smaller than, the pKa values of the carboxylates residues, aspartic acid and glutamic acid in the heteropolymer chain. To date we have used mixtures of guanidinium formate and acetate in equal proportions, with sufficient water to achieve the Gdm+ concentrations 5.36 and 4.39M. Reducing the concentration increases the folding rate without changing the final 100% refolded state. We suggest the slowdown occurs because we have chemically pre-empted the critical links that nucleate the folding process and determine its all-or-nothing character.