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首页> 外文期刊>Biochemistry >A Fluorescent Mutant of the NM Domain of the Yeast Prion Sup35 Provides Insight into Fibril Formation and Stability
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A Fluorescent Mutant of the NM Domain of the Yeast Prion Sup35 Provides Insight into Fibril Formation and Stability

机译：酵母Pri蛋白Sup35 NM域的荧光突变体提供了对原纤维形成和稳定性的了解。

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The Sup35 protein of Saccharomyces cerevisiae forms a prion that generates the [PSIntn] phenotype.nIts NM region governs prion status, forming self-seeding amyloid fibers in vivo and in vitro. A tryptophannmutant of Sup35 (NMF117W) was used to probe its aggregation. Four indicators of aggregation, Trp 117nmaximum emission, Trp polarization, thio-T binding, and light scattering increase, revealed faster aggregationnat 4 u0002C than at 25 u0002C, and all indicators changed in a concerted fashion at the former temperature.nCuriously, at 25 u0002C the changes were not synchronized; the first two indicators, which reflect nucleation,nchanged more quickly than the last two, which reflect fibril formation. These results suggest that nucleation isninsensitive to temperature, whereas fibril extension is temperature dependent. As expected, aggregation isnaccelerated when a small fraction (5%) of the nuclei produced at 4 or 25 u0002C are added to a suspensionncontaining the soluble NM domain, although these nuclei do not seem to propagate any structuralninformation to the growing fibrils. Fibrils grown at 4 u0002C were less stable in GdmCl than those grown atnhigher temperature. However, they were both resistant to high pressure; in fact, both sets of fibrils respondednto high pressure by adopting an altered conformation with a higher capacity for thio-T binding. From thesendata, we calculated the change in volume and free energy associated with this conformational change. AFMnrevealed that the fibrils grown at 4 u0002C were statistically smaller than those grown at 25 u0002C. In conclusion, thenintroduction of Trp 117 allowed us to more carefully dissect the effects of temperature on the aggregation ofnthe Sup35 NM domain.
机译：酿酒酵母的Sup35蛋白形成a病毒，产生[PSIntn]表型。nNM区控制病毒的状态，在体内和体外形成自种的淀粉样纤维。 Sup35的色氨酸突变体（NMF117W）用于探测其聚集。聚集的四个指标，Trp 117n最大发射，Trp极化，硫代-T结合和光散射增加，显示4 u0002C的聚集速度比25 u0002C的要快，并且所有指标在前一个温度下都以一致的方式变化。n奇怪的是，在25 u0002C时。更改未同步；前两个反映成核的指标变化快于后两个反映原纤维形成的指标。这些结果表明成核对温度不敏感，而原纤维延伸是温度依赖性的。如所预期的，当将在4或25 u0002C产生的一小部分核（5％）添加到含有可溶性NM域的悬浮液中时，聚集加速，尽管这些核似乎并未将任何结构信息传播到生长的原纤维。在4 u0002C下生长的原纤维在GdmCl中的稳定性低于在较高温度下生长的原纤维。但是，它们都耐高压。实际上，两组原纤维均通过采用具有更高硫-T结合能力的构象改变而对高压作出反应。根据该数据，我们计算了与该构象变化有关的体积和自由能的变化。 AFMn揭示出，在4u0002℃下生长的原纤维统计上小于在25u0002℃下生长的原纤维。总之，Trp 117的引入使我们可以更仔细地剖析温度对Sup35 NM域聚集的影响。

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