Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland.
Proc Natl Acad Sci U S A. 2013 Dec 10;110(50):20051-6. doi: 10.1073/pnas.1310414110. Epub 2013 Nov 21.
The human protein β2-microglobulin (β2m) aggregates as amyloid fibrils in patients undergoing long-term hemodialysis. Isomerization of Pro32 from its native cis to a nonnative trans conformation is thought to trigger β2m misfolding and subsequent amyloid assembly. To examine this hypothesis, we systematically varied the free-energy profile of proline cis-trans isomerization by replacing Pro32 with a series of 4-fluoroprolines via total chemical synthesis. We show that β2m's stability, (un)folding, and aggregation properties are all influenced by the rate and equilibrium of Pro32 cis-trans isomerization. As anticipated, the β2m monomer was either stabilized or destabilized by respective incorporation of (2S,4S)-fluoroproline, which favors the native cis amide bond, or the stereoisomeric (2S,4R)-fluoroproline, which disfavors this conformation. However, substitution of Pro32 with 4,4-difluoroproline, which has nearly the same cis-trans preference as proline but an enhanced isomerization rate, caused pronounced destabilization of the protein and increased oligomerization at neutral pH. More remarkably, these subtle alterations in chemical composition--incorporation of one or two fluorine atoms into a single proline residue in the 99 amino acid long protein--modulated the aggregation properties of β2m, inducing the formation of polymorphically distinct amyloid fibrils. These results highlight the importance of conformational dynamics for molecular assembly of an amyloid cross-β structure and provide insights into mechanistic aspects of Pro32 cis-trans isomerism in β2m aggregation.
人β2-微球蛋白（β2m）在长期接受血液透析的患者中会聚集形成淀粉样纤维。人们认为脯氨酸 32 从其天然顺式到非天然反式构象的异构化触发了β2m 错误折叠和随后的淀粉样组装。为了检验这一假说，我们通过全化学合成将脯氨酸 32 分别用一系列 4-氟脯氨酸取代，从而系统地改变了脯氨酸顺反异构化的自由能分布。我们表明β2m 的稳定性、（去）折叠和聚集特性都受到 Pro32 顺反异构化的速率和平衡的影响。正如预期的那样，β2m 单体分别被（2S,4S）-氟脯氨酸或其立体异构体（2S,4R）-氟脯氨酸稳定或不稳定，（2S,4S）-氟脯氨酸有利于天然顺酰胺键，而（2S,4R）-氟脯氨酸则不利于这种构象。然而，用 4,4-二氟脯氨酸取代 Pro32，其顺反异构化偏好与脯氨酸几乎相同但异构化速率增强，导致蛋白质明显不稳定并在中性 pH 下增加寡聚化。更显著的是，这些化学组成的细微改变——在 99 个氨基酸长的蛋白质中一个或两个氟原子掺入单个脯氨酸残基——调节了β2m 的聚集特性，诱导形成了多态性不同的淀粉样纤维。这些结果强调了构象动力学对于淀粉样β-交叉结构的分子组装的重要性，并为β2m 聚集中 Pro32 顺反异构化的机制方面提供了新的见解。
