Kinetic intermediates of β(2)-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 β(2)-microglobulin (β(2)-m) at pD=2.5, under which conditions β(2)-m is acid denatured. To study the conformational change in monomeric β(2)-m monitored by NMR spectroscopy, we used (15)N-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 β(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.
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Tsuyoshi
T
Konuma
Eri
E
Chatani
Masanori
M
Yagi
Kazumasa
K
Sakurai
Takahisa
T
Ikegami
Hironobu
H
Naiki
Yuji
Y
Goto
D000682:Amyloid / Q000737:chemistry*; D041961:Deuterium Exchange Measurement / Q000379:methods; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D001613:beta 2-Microglobulin / Q000737:chemistry*
D000682:Amyloid; D001613:beta 2-Microglobulin