Department of Chemistry, East Carolina University, Greenville, NC 27858, USA.
J Mol Biol. 2013 Mar 25;425(6):977-88. doi: 10.1016/j.jmb.2013.01.008. Epub 2013 Jan 11.
The process of transthyretin (TTR) misfolding and aggregation, including amyloid formation, appears to cause a number of degenerative diseases. During amyloid formation, the native protein undergoes a tetramer-to-folded monomer transition, followed by local unfolding of the monomer to an assembly-competent amyloidogenic intermediate. Here we use NMR relaxation dispersion to probe conformational exchange at physiological pH between native monomeric TTR (the F87M/L110M variant) and a small population of a transiently formed amyloidogenic intermediate. The dispersion experiments show that a majority of the residues in the β-sheet containing β-strands D, A, G, and H undergo conformational fluctuations on microsecond-to-millisecond timescales. Exchange broadening is greatest for residues in the outer β-strand H, which hydrogen bonds to β-strand H' of a neighboring subunit in the tetramer, but the associated structural fluctuations propagate across the entire β-sheet. Fluctuations in the other β-sheet are limited to the outer β-strand F, which packs against strand F' in the tetramer, while the B, C, and E β-strands of this sheet remain stable. The structural changes were also investigated under more forcing amyloidogenic conditions (pH6.4-3.7), where β-strand D and regions of the D-E and E-F loops were additionally destabilized, increasing the population of the amyloidogenic intermediate and accelerating amyloid formation. Strands B, C, and E appear to maintain native-like conformations in the partially unfolded, amyloidogenic state of wild-type TTR. In the case of the protective mutant T119M, the conformational fluctuations are suppressed under both physiological and mildly acidic conditions, indicating that the dynamic properties of TTR correlate well with its aggregation propensity.
转甲状腺素蛋白（TTR）错误折叠和聚集的过程，包括淀粉样纤维的形成，似乎导致了许多退行性疾病。在淀粉样纤维形成过程中，天然蛋白质经历四聚体到折叠单体的转变，随后单体局部展开到组装能力的淀粉样纤维中间态。在这里，我们使用 NMR 弛豫色散来探测生理 pH 下天然单体 TTR（F87M/L110M 变体）和一小部分瞬态形成的淀粉样纤维中间态之间的构象交换。色散实验表明，β-片层中包含的β-链 D、A、G 和 H 的大多数残基在微秒到毫秒的时间尺度上经历构象波动。β-链 H 中与四聚体中相邻亚基的β-链 H'形成氢键的残基的交换展宽最大，但相关的结构波动会传播到整个β-片层。其他β-片层的波动仅限于与四聚体中β-链 F'相互作用的β-链 F，而该β-片层的 B、C 和 E β-链则保持稳定。还在更具淀粉样纤维形成倾向的条件（pH6.4-3.7）下研究了这些结构变化，其中β-链 D 以及 D-E 和 E-F 环的区域也被进一步去稳定化，增加了淀粉样纤维中间态的丰度并加速了淀粉样纤维的形成。在野生型 TTR 的部分展开的淀粉样纤维形成状态下，β-链 B、C 和 E 似乎保持了类似天然的构象。对于保护性突变体 T119M，在生理和轻度酸性条件下，构象波动都受到抑制，这表明 TTR 的动态特性与其聚集倾向密切相关。
