Fibril Growth Kinetics Reveal a Region of β2-microglobulin Important for Nucleation and Elongation of Aggregation

Abstract

Amyloid is a highly ordered form of aggregate comprising long, straight and unbranched proteinaceous fibrils that are formed with characteristic nucleation-dependent kinetics in vitro. Currently, the structural molecular mechanism of fibril nucleation and elongation is poorly understood. Here, we investigate the role of the sequence and structure of the initial monomeric precursor in determining the rates of nucleation and elongation of human β2-microglobulin (β2m). We describe the kinetics of seeded and spontaneous (unseeded) fibril growth of wild-type β2m and 12 variants at pH 2.5, targeting specifically an aromatic-rich region of the polypeptide chain (residues 62–70) that has been predicted to be highly amyloidogenic. The results reveal the importance of aromatic residues in this part of the β2m sequence in fibril formation under the conditions explored and show that this region of the polypeptide chain is involved in both the nucleation and the elongation phases of fibril formation. Structural analysis of the conformational properties of the unfolded monomer for each variant using NMR relaxation methods revealed that all variants contain significant non-random structure involving two hydrophobic clusters comprising regions 29–51 and 58–79, the extent of which is critically dependent on the sequence. No direct correlation was observed, however, between the extent of non-random structure in the unfolded state and the rates of fibril nucleation and elongation, suggesting that the early stages of aggregation involve significant conformational changes from the initial unfolded state. Together, the data suggest a model for β2m amyloid formation in which structurally specific interactions involving the highly hydrophobic and aromatic-rich region comprising residues 62–70 provide a complementary interface that is key to the generation of amyloid fibrils for this protein at acidic pH.