Volume Exclusion and Soft Interaction Effects on Protein Stability under Crowded Conditions
Volume Exclusion and Soft Interaction Effects on Protein Stability under Crowded Conditions
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DOI：
10.1021/bi100727y
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发表时间：
2010-08-24
期刊：
影响因子：
2.9
通讯作者：
Pielak, Gary J.
中科院分区：
文献类型：
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作者：
Miklos, Andrew C.;Li, Conggang;Pielak, Gary J.
大多数蛋白质在自然界中在拥挤的条件下发挥作用，而拥挤会改变蛋白质的特性。然而，拥挤效应的量化很困难，因为每升含有数百克大分子的溶液通常会干扰所研究蛋白质的观察。大分子拥挤模型倾向于关注拥挤剂的空间效应，忽略拥挤剂和测试蛋白质之间潜在的化学相互作用。在这里，我们报告了第一个系统的、定量的、残留水平的拥挤效应对球状蛋白平衡稳定性的研究。我们使用包含不同分子量的聚（乙烯基吡咯烷酮）（PVP）作为拥挤剂和胰凝乳蛋白酶抑制剂2（CI2）作为小球状测试蛋白的系统。通过 NMR 检测的酰胺交换来量化稳定性。我们从硬粒子排除、限制和软相互作用方面分析了数据。对于所有拥挤的条件，几乎所有观察到的残留物都会产生稳定作用。例外情况是稳定性未改变的残基。在 PVP 浓度为 100 g/L 时，数据与硬颗粒排除理论一致。在较高浓度下，数据与限制更加一致。数据显示，拥挤器还通过弱结合其天然状态来稳定测试蛋白。我们得出的结论是，在将理论和实验应用于大分子拥挤的研究时，需要认真考虑自然态结合和其他软相互作用的作用。
Most proteins function in nature under crowded conditions, and crowding can change protein properties. Quantification of crowding effects, however, is difficult because solutions containing hundreds of grams of macromolecules per liter often interfere with the observation of the protein being studied. Models for macromolecular crowding tend to focus on the steric effects of crowders, neglecting potential chemical interactions between the crowder and the test protein. Here, we report the first systematic, quantitative, residue-level study of crowding effects on the equilibrium stability of a globular protein. We used a system comprising poly(vinylpyrrolidone)s (PVPs) of varying molecular weights as crowding agents and chymotrypsin inhibitor 2 (CI2) as a small globular test protein. Stability was quantified with NMR-detected amide exchange. We analyzed the data in terms of hard particle exclusion, confinement, and soft interactions. For all crowded conditions, nearly every observed residue experiences a stabilizing effect. The exceptions are residues for which stabilities are unchanged. At a PVP concentration of 100 g/L, the data are consistent with theories of hard particle exclusion. At higher concentrations, the data are more consistent with confinement. The data show that the crowder also stabilizes the test protein by weakly binding its native state. We conclude that the role of native-state binding and other soft interactions needs to be seriously considered when applying both theory and experiment to studies of macromolecular crowding.