Walters Robert H, Murphy Regina M
Department of Chemical and Biological Engineering, University of Wisconsin, Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
J Mol Biol. 2009 Nov 6;393(4):978-92. doi: 10.1016/j.jmb.2009.08.034. Epub 2009 Aug 20.
Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, of which the best known is Huntington's. Expansion of the polyglutamine domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. The age of onset of disease is inversely correlated with the length of the polyglutamine domain; this correlation motivates an examination of the role of the length of the domain on aggregation. In this investigation, peptides containing 8 to 24 glutamines were synthesized, and their conformational and aggregation properties were examined. All peptides lacked secondary structure. Fluorescence resonance energy transfer studies revealed that the peptides became increasingly collapsed as the number of glutamine residues increased. The effective persistence length was estimated to decrease from approximately 11 to approximately 7 A as the number of glutamines increased from 8 to 24. A comparison of our data with theoretical results suggests that phosphate-buffered saline is a good solvent for Q8 and Q12, a theta solvent for Q16, and a poor solvent for Q20 and Q24. By dynamic light scattering, we observed that Q16, Q20, and Q24, but not Q8 or Q12, immediately formed soluble aggregates upon dilution into phosphate-buffered saline at 37 degrees C. Thus, Q16 stands at the transition point between good and poor solvent and between stable and aggregation-prone peptide. Examination of aggregates by transmission electron microscopy, along with kinetic assays for sedimentation, provided evidence indicating that soluble aggregates mature into sedimentable aggregates. Together, the data support a mechanism of aggregation in which monomer collapse is accompanied by formation of soluble oligomers; these soluble species lack regular secondary structure but appear morphologically similar to the sedimentable aggregates into which they eventually mature.
蛋白质中异常扩展的聚谷氨酰胺结构域与多种神经退行性疾病相关，其中最广为人知的是亨廷顿舞蹈症。聚谷氨酰胺结构域的扩展促进了受影响蛋白质的聚集，并且多项研究直接将聚集与神经毒性联系起来。疾病的发病年龄与聚谷氨酰胺结构域的长度呈负相关；这种相关性促使人们研究该结构域长度对聚集的作用。在本研究中，合成了含有8至24个谷氨酰胺的肽段，并检测了它们的构象和聚集特性。所有肽段均缺乏二级结构。荧光共振能量转移研究表明，随着谷氨酰胺残基数量的增加，肽段变得越来越紧凑。当谷氨酰胺数量从8增加到24时，有效持久长度估计从约11埃减少到约7埃。将我们的数据与理论结果进行比较表明，磷酸缓冲盐水对Q8和Q12是良溶剂，对Q16是θ溶剂，对Q20和Q24是不良溶剂。通过动态光散射，我们观察到，在37℃下稀释到磷酸缓冲盐水中时，Q16、Q20和Q24会立即形成可溶性聚集体，而Q8或Q12则不会。因此，Q16处于良溶剂与不良溶剂之间以及稳定肽段与易于聚集的肽段之间的转变点。通过透射电子显微镜检查聚集体，并进行沉降动力学测定，提供了证据表明可溶性聚集体会成熟为可沉降聚集体。总之，这些数据支持一种聚集机制，即单体紧凑化伴随着可溶性寡聚体的形成；这些可溶性物种缺乏规则的二级结构，但在形态上与它们最终成熟形成的可沉降聚集体相似。
