Lysine carbamoylation during urea denaturation remodels the energy landscape of human transthyretin dissociation linked to unfolding.

Abstract

Chemical denaturants such as urea have become indispensable in modern protein science for measuring the energetics of protein folding and assembly. Denaturants bind to and preferentially stabilize denatured states, folding transition states, and folding intermediates over the native state, allowing experimental access to free energies of folding and insights into folding mechanisms. However, too little attention is paid to the established chemical instability of aqueous urea, that is, its decomposition into the reactive electrophile ammonium cyanate or isocyanic acid depending on the solution pH. Protein carbamoylation by cyanate/isocyanic acid can change the dissociation and/or unfolding free energy landscape of the protein under study with time. This problem is exemplified using the human blood protein transthyretin (TTR), a kinetically stable transporter of thyroid hormone and holo-retinol binding protein. The dissociation, misfolding, and aggregation of TTR are associated with a prominent human amyloid disease. We demonstrate that modification of TTR by cyanate reshapes the energy landscape of TTR tetramer dissociation and unfolding on multiple time scales. Like certain halide anions and the more chemically inert thiocyanate anion, cyanate binds weakly and non-covalently to the thyroid hormone binding interface in the TTR tetramer. The close proximity of the bound cyanate ion to the pK_a-perturbed lysine 15 ε-amino side chain nucleophile in the thyroid hormone binding sites of TTR favors carbamoylation of this nitrogen. Lysine 15 ε-amino carbamoylation substantially slows down TTR tetramer dissociation mediated by urea denaturation, thus introducing kinetic heterogeneity early in the unfolding reaction. Slower carbamoylation of the subpopulation of other, less pK_a-perturbed lysine ε-amino groups hastens tetramer unfolding, leading to non-exponential, sigmoidal unfolding trajectories. We thus demonstrate that lysine carbamoylation in urea solutions can strongly alter protein unfolding energetics and the mechanism of unfolding.