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

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• 作者：Tsuyoshi Konuma¹ ; ^† ; ; Eri Chatani¹ ; ^† ; ; ¹ ; Masanori Yagi¹ ; Kazumasa Sakurai¹ ; Takahisa Ikegami¹ ; Hironobu Naiki² ; Yuji Goto¹ ; ^{ygoto@protein.osaka-u.ac.jp}
• 关键词：protein misfolding ; intermediate of fibril elongation ; dialysis-related amyloidosis ; pulse-labeling H/D exchange ; NMR spectroscopy
• 刊名：Journal of Molecular Biology
• 出版年：2011
• 出版时间：21 January 2011
• 年：2011
• 卷：405
• 期：3
• 页码：851-862
• 全文大小：1372 K

文摘

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.