Aggregation of a slow-folding mutant of a β-clam protein proceeds through a monomeric nucleus
Aggregation of a slow-folding mutant of a β-clam protein proceeds through a monomeric nucleus
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DOI：
10.1021/bi047404e
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发表时间：
2005-05-17
期刊：
影响因子：
2.9
通讯作者：
Gierasch, LM
中科院分区：
文献类型：
--
作者：
Ignatova, Z;Gierasch, LM
了解蛋白质聚集的机制，导致结构淀粉样原纤维或无定形包涵体样沉积物，应该有助于确定潜在的治疗干预策略，以治疗破坏性的淀粉样疾病。在这里，我们重点研究了β -蛤蛋白慢折叠突变体的体外聚集，细胞维甲酸结合蛋白I (P39A CRABP I)，当在大肠杆菌中表达时形成包涵体。通过观察基于荧光素的双砷染料（FlAsH）的荧光来监测聚集，该染料连接到一个四碳基基，在这里并入一个灵活的omega环。FlAsH荧光信号对含有四合基cys的P39A CRABP I是天然蛋白还是未折叠蛋白敏感，并与其他技术结合使用以跟踪聚集形成。聚合过程符合核依赖聚合模型，详细的动力学分析表明，能量不利的核是单体的。先前对聚（Gln）物种也得出了类似的结论[Chen， S., Ferrone， F. A., and Wetzel， R. (2002) Proc. Natl。学会科学。[美国文献99,11884-11889]并指出错误折叠的中间体和大量单体之间的不利平衡是导致聚集的原因。与野生型相比，P39A突变去除了螺旋停止信号，可能会减慢P39A CRABP I中β -桶的关闭速度，使其易于聚集。广角x射线散射结果表明，P39A CRABP I中间体形成的非晶态聚集体主要含有层状结构的β链，相邻β链之间的间距为10.03埃。
Mechanistic understanding of protein aggregation, leading either to structured amyloid fibrils or to amorphous inclusion body-like deposits, should facilitate the identification of potential therapeutic intervention strategies for the devastating amyloid-based diseases. Here we focus on the in vitro aggregation of a slow-folding mutant of the beta-clam protein, cellular retinoic acid-binding protein I (P39A CRABP I), which forms inclusion bodies when expressed in Escherichia coli. Aggregation was monitored by observing the fluorescence of a fluorescein-based biarsenical dye (FlAsH) that ligates to a tetra-Cys motif, here incorporated into a flexible Omega-loop. The fluorescence signal of FlAsH on the tetra-Cys-containing P39A CRABP I is sensitive to whether this protein is native or unfolded, and was used in combination with other techniques to follow aggregate formation. The aggregation time course is compatible with a nucleation-dependent polymerization model, and detailed kinetic analysis showed that the energetically unfavorable nucleus is monomeric. A similar conclusion was reached previously for poly(Gln) species [Chen, S., Ferrone, F. A., and Wetzel, R. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11884-11889] and points to an unfavorable equilibrium between the misfolded intermediate and the bulk pool of monomers as causative in aggregation. The P39A mutation, which removes a helix-stop signal, may slow closure of the beta-barrel in P39A CRABP I relative to the wild type, leaving it vulnerable to aggregation. Wide-angle X-ray scattering showed that the amorphous aggregates formed by the aggregation-prone intermediates of P39A CRABP I contain predominantly beta-strands structured in a lamellar fashion with 10.03 angstrom spacing between adjacent beta-sheets.