Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037.
Skaggs Institute of Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 2018 Jul 3;115(27):E6201-E6208. doi: 10.1073/pnas.1807024115. Epub 2018 Jun 18.
Aggregation of transthyretin (TTR) is the causative agent for TTR cardiomyopathy and polyneuropathy amyloidoses. Aggregation is initiated by dissociation of the TTR tetramer into a monomeric intermediate, which self-assembles into amyloid. The coupled multiple-step equilibria and low-concentration, aggregation-prone intermediates are challenging to probe using conventional assays. We report a F-NMR assay that leverages a highly sensitive trifluoroacetyl probe at a strategic site that gives distinct F chemical shifts for the TTR tetramer and monomeric intermediate and enables direct quantification of their populations during the aggregation process. Integration of real-time F-NMR and turbidity measurements as a function of temperature allows kinetic and mechanistic dissection of the aggregation pathway of both wild-type and mutant TTR. At physiological temperature, the monomeric intermediate formed by wild-type TTR under mildly acidic conditions rapidly aggregates into species that are invisible to NMR, leading to loss of the NMR signal at the same rate as the turbidity increase. Lower temperature accelerates tetramer dissociation and decelerates monomer tetramerization and oligomerization via reduced hydrophobic interactions associated with packing of a phenylalanine (F87) into a neighboring protomer. As a result, the intermediate accumulates to a higher level, and formation of higher-order aggregates is delayed. Application of this assay to pathogenic (V30M, L55P, and V122I) and protective (T119M) mutants revealed significant differences in behavior. A monomeric intermediate was observed only for V122I: aggregation of V30M and L55P proceeds without an observable monomeric intermediate, whereas the protective mutant T119M remains resistant to tetramer dissociation and aggregation.
转甲状腺素蛋白（TTR）的聚集是 TTR 心肌病和多发性神经病淀粉样变的原因。聚集是由 TTR 四聚体解离为单体中间物引发的，该中间物自组装成淀粉样蛋白。偶联的多步平衡和低浓度、易于聚集的中间物难以用常规方法进行探测。我们报告了一种 F-NMR 测定法，该方法利用在一个战略位置上的高灵敏度三氟乙酰基探针，该探针为 TTR 四聚体和单体中间物提供了独特的 F 化学位移，并能够在聚集过程中直接定量它们的种群。将实时 F-NMR 和浊度测量作为温度的函数进行集成，允许对野生型和突变型 TTR 的聚集途径进行动力学和机制剖析。在生理温度下，在轻度酸性条件下形成的野生型 TTR 的单体中间物迅速聚集成无法用 NMR 检测到的物种，导致 NMR 信号以与浊度增加相同的速率丢失。较低的温度通过降低与苯丙氨酸（F87）包装到相邻原聚体相关的疏水力，加速四聚体解离并减慢单体四聚化和寡聚化。因此，中间物积累到更高的水平，并且更高阶的聚集体形成被延迟。该测定法应用于致病性（V30M、L55P 和 V122I）和保护性（T119M）突变体，揭示了它们行为的显著差异。仅观察到 V122I 的单体中间物：V30M 和 L55P 的聚集没有可观察到的单体中间物，而保护性突变体 T119M 仍然抵抗四聚体解离和聚集。
