Residue level quantification of protein stability in living cells
Residue level quantification of protein stability in living cells
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DOI：
10.1073/pnas.1406845111
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发表时间：
2014-08-05
影响因子：
11.1
通讯作者：
Pielak, Gary J.
中科院分区：
文献类型：
--
作者：
Monteith, William B.;Pielak, Gary J.
细胞内环境不同于进行大多数生物物理和生物化学研究的稀释条件。这种差异使得实验学家和理论家都致力于解决理解细胞内环境如何影响生物聚合物性质的挑战性任务。尽管越来越多的细胞内研究，有一个缺乏定量的，残留水平的信息平衡热力学蛋白质的稳定性在非扰动条件下。我们报告使用核磁共振检测的氢氘交换淬灭细胞裂解物，以测量个人的开放自由能的56-aa的B1结构域的蛋白G（GB 1）在活大肠杆菌细胞，而不添加不稳定的共溶质或热。与稀溶液数据（pH 7.6和37 ℃）的比较表明，细胞中的开放自由能增加了1.14 +/- 0.05 kcal/mol。重要的是，我们还表明，同质蛋白质拥挤使GB 1不稳定，突出了重建细胞内部的挑战。我们讨论了我们的研究结果在硬核排除体积效应，电荷-电荷GB 1拥挤的相互作用，和其他因素。淬灭裂解物的方法确定的残留物最重要的折叠GB 1在细胞中，并应证明有用的定量细胞中的其他球状蛋白质的稳定性，以获得更全面的了解细胞内环境对蛋白质化学的影响。
The intracellular milieu differs from the dilute conditions in which most biophysical and biochemical studies are performed. This difference has led both experimentalists and theoreticians to tackle the challenging task of understanding how the intracellular environment affects the properties of biopolymers. Despite a growing number of in-cell studies, there is a lack of quantitative, residue-level information about equilibrium thermodynamic protein stability under nonperturbing conditions. We report the use of NMR-detected hydrogen-deuterium exchange of quenched cell lysates to measure individual opening free energies of the 56-aa B1 domain of protein G (GB1) in living Escherichia coli cells without adding destabilizing cosolutes or heat. Comparisons to dilute solution data (pH 7.6 and 37 degrees C) show that opening free energies increase by as much as 1.14 +/- 0.05 kcal/mol in cells. Importantly, we also show that homogeneous protein crowders destabilize GB1, highlighting the challenge of recreating the cellular interior. We discuss our findings in terms of hard-core excluded volume effects, charge-charge GB1-crowder interactions, and other factors. The quenched lysate method identifies the residues most important for folding GB1 in cells, and should prove useful for quantifying the stability of other globular proteins in cells to gain a more complete understanding of the effects of the intracellular environment on protein chemistry.