The residual structure of acid-denatured ß2 -microglobulin is relevant to an ordered fibril morphology.

ß2 -Microglobulin (ß2m) forms amyloid fibrils in vitro under acidic conditions. Under these conditions, the residual structure of acid-denatured ß2m is relevant to seeding and fibril extension processes. Disulfide (SS) bond-oxidized ß2m has been shown to form rigid, ordered fibrils, whereas SS bond-reduced ß2m forms curvy, less-ordered fibrils. These findings suggest that the presence of an SS bond affects the residual structure of the monomer, which subsequently influences the fibril morphology. To clarify this process, we herein performed NMR experiments. The results obtained revealed that oxidized ß2m contained a residual structure throughout the molecule, including the N- and C-termini, whereas the residual structure of the reduced form was localized and other regions had a random coil structure. The range of the residual structure in the oxidized form was wider than that of the fibril core. These results indicate that acid-denatured ß2m has variable conformations. Most conformations in the ensemble cannot participate in fibril formation because their core residues are hidden by residual structures. However, when hydrophobic residues are exposed, polypeptides competently form an ordered fibril. This conformational selection phase may be needed for the ordered assembly of amyloid fibrils.

Enhanced accessibility and hydrophobicity of amyloidogenic intermediates of the ß2-microglobulin D76N mutant revealed by high-pressure experiments.

ß2-Microglobulin (ß2m) is the causative protein of dialysis-related amyloidosis. Its unfolding mainly proceeds along the pathway of NC →UC ⇄ UT, whereas refolding follows the UT → IT (→NT) →NC 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 IT state is a putative amyloidogenic precursor state. Several aggregation-prone variants of ß2m have been reported to date. One of these variants is D76N ß2m, 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 IT state for D76N in the refolding process. In addition, the IT 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.

Loosening of Side-Chain Packing Associated with Perturbations in Peripheral Dynamics Induced by the D76N Mutation of ß2-Microglobulin Revealed by Pressure-NMR and Molecular Dynamic Simulations.

ß2-Microglobulin (ß2m) is the causative protein of dialysis-related amyloidosis, and its D76N variant is less stable and more prone to aggregation. Since their crystal structures are indistinguishable from each other, enhanced amyloidogenicity induced by the mutation may be attributed to changes in the structural dynamics of the molecule. We examined pressure and mutation effects on the ß2m molecule by NMR and MD simulations, and found that the mutation induced the loosening of the inter-sheet packing of ß2m, which is relevant to destabilization and subsequent amyloidogenicity. On the other hand, this loosening was coupled with perturbed dynamics at some peripheral regions. The key result for this conclusion was that both the mutation and pressure induced similar reductions in the mobility of these residues, suggesting that there is a common mechanism underlying the suppression of inherent fluctuations in the ß2m molecule. Analyses of data obtained under high pressure conditions suggested that the network of dynamically correlated residues included not only the mutation site, but also distal residues, such as those of the C- and D-strands. Reductions in these local dynamics correlated with the loosening of inter-sheet packing.