Enhanced accessibility and hydrophobicity of amyloidogenic intermediates of the β2-microglobulin D76N mutant revealed by high-pressure experiments

β2-Microglobulin (β2m) is the causative protein of dialysis-related amyloidosis. Its unfolding mainly proceeds along the pathway of N_C →U_C ⇄ U_T, whereas refolding follows the U_T → I_T (→N_T) →N_C pathway, in which N, I, and U are the native, intermediate, and unfolded states, respectively, with the Pro32 peptidyl-prolyl bond in cis or trans conformation as indicated by the subscript. It is noted that the I_T state is a putative amyloidogenic precursor state. Several aggregation-prone variants of β2m have been reported to date. One of these variants is D76N β2m, which is a naturally occurring amyloidogenic mutant. To elucidate the molecular mechanisms contributing to the enhanced amyloidogenicity of the mutant, we investigated the equilibrium and kinetic transitions of pressure-induced folding/unfolding equilibria in the wild type and D76N mutant by monitoring intrinsic tryptophan and 1-anilino-8-naphthalene sulfonate fluorescence. An analysis of kinetic data revealed that the different folding/unfolding behaviors of the wild type and D76N mutant were due to differences in the activation energy between the unfolded and the intermediate states as well as stability of the native state, leading to more rapid accumulation of I_T state for D76N in the refolding process. In addition, the I_T state was found to assume more hydrophobic nature. These changes induced the enhanced amyloidogenicity of the D76N mutant and the distinct pathogenic symptoms of patients. Our results suggest that the stabilization of the native state will be an effective approach for suppressing amyloid fibril formation of this mutant.