Biophysical Chemistry, Department of Chemistry, University of Konstanz, Konstanz, Germany; Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
Molecular Microbiology, Department of Biology, University of Konstanz, Konstanz, Germany; Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
Biophys J. 2018 Apr 24;114(8):1847-1857. doi: 10.1016/j.bpj.2018.02.037.
Polyglutamine (polyQ) diseases, including Huntington's disease, result from the aggregation of an abnormally expanded polyQ repeat in the affected protein. The length of the polyQ repeat is essential for the disease's onset; however, the molecular mechanism of polyQ aggregation is still poorly understood. Controlled conditions and initiation of the aggregation process are prerequisites for the detection of transient intermediate states. We present an attenuated total reflection Fourier-transform infrared spectroscopic approach combined with protein immobilization to study polyQ aggregation dependent on the polyQ length. PolyQ proteins were engineered mimicking the mammalian N-terminus fragment of the Huntingtin protein and containing a polyQ sequence with the number of glutamines below (Q11), close to (Q38), and above (Q56) the disease threshold. A monolayer of the polyQ construct was chemically immobilized on the internal reflection element of the attenuated total reflection cell, and the aggregation was initiated via enzymatic cleavage. Structural changes of the polyQ sequence were monitored by time-resolved infrared difference spectroscopy. We observed faster aggregation kinetics for the longer sequences, and furthermore, we could distinguish β-structured intermediates for the different constructs, allowing us to propose aggregation mechanisms dependent on the repeat length. Q11 forms a β-structured aggregate by intermolecular interaction of stretched monomers, whereas Q38 and Q56 undergo conformational changes to various β-structured intermediates, including intramolecular β-sheets.
多聚谷氨酰胺（polyQ）疾病，包括亨廷顿病，是由受影响蛋白中异常扩展的多聚 Q 重复序列的聚集引起的。多聚 Q 重复序列的长度对疾病的发作至关重要；然而，多聚 Q 聚集的分子机制仍知之甚少。受控条件和聚集过程的启动是检测瞬态中间状态的前提。我们提出了一种衰减全反射傅里叶变换红外光谱方法，结合蛋白质固定化，研究依赖于多聚 Q 长度的多聚 Q 聚集。多聚 Q 蛋白通过模拟亨廷顿蛋白的哺乳动物 N 端片段进行工程设计，并包含一个多聚 Q 序列，其谷氨酰胺数量低于（Q11）、接近（Q38）和高于（Q56）疾病阈值。多聚 Q 构建体的单层通过化学固定在衰减全反射池的内部反射元件上，并通过酶切启动聚合。通过时间分辨红外差光谱监测多聚 Q 序列的结构变化。我们观察到较长序列的聚合动力学更快，此外，我们还可以区分不同构建体的β-结构中间体，使我们能够提出依赖于重复长度的聚合机制。Q11 通过伸展单体的分子间相互作用形成β-结构聚集体，而 Q38 和 Q56 经历构象变化为各种β-结构中间体，包括分子内β-片层。
