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 beta(2)-microglobulin (beta(2)-m) at pD = 2.5, under which conditions beta(2)-m is acid denatured. To study the conformational change in monomeric beta(2)-m monitored by NMR spectroscopy, we used N-15-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 beta(2)-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. (C) 2010 Elsevier Ltd. All rights reserved.