Sensitivity-enhanced NMR reveals alterations in protein structure by cellular milieus.
Sensitivity-enhanced NMR reveals alterations in protein structure by cellular milieus.
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DOI：
10.1016/j.cell.2015.09.024
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发表时间：
2015-10-22
期刊：
影响因子：
64.5
通讯作者：
Lindquist S
中科院分区：
文献类型：
--
作者：
Frederick KK;Michaelis VK;Corzilius B;Ong TC;Jacavone AC;Griffin RG;Lindquist S
生物过程发生在包含无数潜在相互作用的复杂环境中。不幸的是，生物物理技术灵敏度的限制通常限制了对纯化系统的结构研究，其浓度高于内源性水平的数量级。动态核极化（DNP）可以极大地提高核磁共振光谱的灵敏度，使生物复杂环境中的结构研究成为可能。在这里，我们应用DNP NMR研究了酵母朊蛋白Sup35的结构，该蛋白既包含环境敏感折叠途径，也包含内在无序区域。我们将外源制备的同位素标记蛋白添加到氘化裂解物中，使生物环境“隐形”，并实现DNP的高效极化转移。在这种环境下，结构变化发生在已知影响生物活性的区域，但在纯化样品中本质上是无序的。因此，DNP使内源性水平的蛋白质结构研究在生物环境中成为可能，而这些环境可以影响蛋白质结构。
Biological processes occur in complex environments containing a myriad of potential interactors. Unfortunately, limitations on the sensitivity of biophysical techniques normally restrict structural investigations to purified systems, at concentrations that are orders of magnitude above endogenous levels. Dynamic nuclear polarization (DNP) can dramatically enhance the sensitivity of NMR spectroscopy and enable structural studies in biologically complex environments. Here we applied DNP NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an instrinsically disordered region, the yeast prion protein Sup35. We added an exogenously-prepared isotopically-labeled protein to deuterated lysates, rendering the biological environment “invisible” and enabling highly efficient polarization transfer for DNP. In this environment, structural changes occurred in a region known to influence biological activity but intrinsically disordered in purified samples. Thus, DNP makes structural studies of proteins at endogenous levels in biological contexts possible and such contexts can influence protein structure.