Kinetic Intermediates of β2-Microglobulin Fibril Elongation Probed by Pulse-Labeling H/D Exchange Combined with NMR Analysis

Amyloid fibril elongation in denatured proteins involves cycles of coupled binding and misfolding. To gain insights into possible kinetic intermediates, we performed hydrogen/deuterium exchange of amide protons during fibril elongation with β2-microglobulin (β2-m) at pD = 2.5, under which conditions β2-m is acid denatured. To study the conformational change in monomeric β2-m monitored by NMR spectroscopy, we used 15N-labeled monomers and nonlabeled seeds. Pulse-labeling hydrogen/deuterium exchange with a quenched-flow apparatus indicated that the rate-limiting intermediate at pD = 2.5 is not protected from the exchange, even disrupting a hydrophobic cluster present in the acid-denatured β2-m. Significant protection was acquired upon transition to the fibrils. In view of the suggestion that the rate-limiting intermediates are bound to the lateral surface of seed fibrils, weak interactions with a largely unfolded conformation might be useful for their dynamic sliding to the growing ends. The results support a new model of fibril elongation with intermediates bound to the lateral surface of seeds.