An optimized strategy to measure protein stability highlights differences between cold and hot unfolded states.
An optimized strategy to measure protein stability highlights differences between cold and hot unfolded states.
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DOI：
10.1038/ncomms15428
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发表时间：
2017-05-18
影响因子：
16.6
通讯作者：
Temussi PA
中科院分区：
文献类型：
--
作者：
Alfano C;Sanfelice D;Martin SR;Pastore A;Temussi PA
大分子拥挤应该通过排除体积效应来稳定蛋白质的折叠形式。这种解释受到质疑，观察到的效应归因于与其他细胞成分的弱相互作用。在这里，我们得出结论，蛋白质的稳定性受到体积排斥的影响，当群体的大小更接近所研究的蛋白质时，这种影响更为明显。通过选择酵母卵黄蛋白（一种在零度以上经历冷变性的蛋白质），可以准确地评估体积排除效应，因为在低温下展开的形式比在高温下展开的形式更大。为了达到对稳定性变化的最佳灵敏度，我们引入了一个从稳定性曲线导出的经验参数。根据Privalov对冷变性的解释，PEG 20对冷变性的巨大影响可以通过水活度的变化来解释。拥挤效应——在考虑细胞环境时很重要——极大地影响蛋白质的稳定性。本文研究了大分子挤压剂对高低温蛋白展开的影响，发现当蛋白质和挤压剂的体积相似时，体积排斥效应更大。
Macromolecular crowding ought to stabilize folded forms of proteins, through an excluded volume effect. This explanation has been questioned and observed effects attributed to weak interactions with other cell components. Here we show conclusively that protein stability is affected by volume exclusion and that the effect is more pronounced when the crowder's size is closer to that of the protein under study. Accurate evaluation of the volume exclusion effect is made possible by the choice of yeast frataxin, a protein that undergoes cold denaturation above zero degrees, because the unfolded form at low temperature is more expanded than the corresponding one at high temperature. To achieve optimum sensitivity to changes in stability we introduce an empirical parameter derived from the stability curve. The large effect of PEG 20 on cold denaturation can be explained by a change in water activity, according to Privalov's interpretation of cold denaturation. Crowding effects—important when considering cellular environments—greatly influence protein stability. Here the authors study the impact of macromolecular crowders on high and low temperature protein unfolding, and show that volume exclusion effects are larger when the protein and crowder volumes are similar.