Development of in vivo HDX-MS with applications to a TonB-dependent transporter and other proteins.
Development of in vivo HDX-MS with applications to a TonB-dependent transporter and other proteins.
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DOI：
10.1002/pro.4402
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发表时间：
2022-09
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影响因子：
--
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氢-重离子交换质谱仪(HDX-MS)是监测溶液中蛋白质动力学的有力工具。然而，HDX标记的可逆性在很大程度上限制了其在体外系统中的应用。在这里，我们描述了一种在活的大肠杆菌细胞中测量HDX-MS的方法，该方法应用于BtuB，一种存在于外膜(OMs)中的TonB依赖的转运蛋白。BtuB是一种在体内测试HDX-MS的方便和生物学有趣的系统，因为它的HDX行为是可控的，并且在B12转运周期中发生了大量的结构重排。我们之前在天然OMS中进行的HDX-MS研究为B12结合和断裂一种名为离子锁的盐桥提供了证据，这一事件导致氨基末端的展开。尽管纯化的OM提供了比重组系统更自然的环境，但在裂解过程中细胞被膜的破坏扰乱了BtuB和驱动B12运输的TonB复合体之间的联系。BtuB的插入域(BtuBp)对B12结合的体内HDX反应证实了我们之前的体外发现，即B12本身就足以打破离子锁。此外，我们仍然没有发现B12结合诱导的BtuBp其他区域能够使B12通过的证据。我们的方法成功地报告了在同一测量中测量的几种内源性大肠杆菌蛋白的HDX。我们在活细胞中进行HDX的成功为未来在天然细胞环境中进行HDX-MS研究打开了可能性。我们提出了一种在体内进行HDX-MS的方案，重点是BtuB，一种天然的膜环境被认为对B12运输具有重要机械作用的蛋白质。体内HDX-MS数据证实了我们之前对B12结合引发变构的体外HDX-MS研究的结论。我们在BtuB和其他蛋白质方面的成功为在天然细胞环境中进行更多的HDX-MS研究打开了可能性。
Hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) is a powerful tool that monitors protein dynamics in solution. However, the reversible nature of HDX labels has largely limited the application to in vitro systems. Here, we describe a protocol for measuring HDX‐MS in living Escherichia coli cells applied to BtuB, a TonB‐dependent transporter found in outer membranes (OMs). BtuB is a convenient and biologically interesting system for testing in vivo HDX‐MS due to its controllable HDX behavior and large structural rearrangements that occur during the B12 transport cycle. Our previous HDX‐MS study in native OMs provided evidence for B12 binding and breaking of a salt bridge termed the Ionic Lock, an event that leads to the unfolding of the amino terminus. Although purified OMs provide a more native‐like environment than reconstituted systems, disruption of the cell envelope during lysis perturbs the linkage between BtuB and the TonB complex that drives B12 transport. The in vivo HDX response of BtuB's plug domain (BtuBp) to B12 binding corroborates our previous in vitro findings that B12 alone is sufficient to break the Ionic Lock. In addition, we still find no evidence of B12 binding‐induced unfolding in other regions of BtuBp that could enable B12 passage. Our protocol was successful in reporting on the HDX of several endogenous E. coli proteins measured in the same measurement. Our success in performing HDX in live cells opens the possibility for future HDX‐MS studies in a native cellular environment. We present a protocol for performing in vivo HDX‐MS, focusing on BtuB, a protein whose native membrane environment is believed to be mechanistically important for B12 transport. The in vivo HDX‐MS data corroborate the conclusions from our previous in vitro HDX‐MS study of the allostery initiated by B12 binding. Our success with BtuB and other proteins opens the possibility for performing additional HDX‐MS studies in a native cellular environment.
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影响因子：
3.2
作者：
Gresock, Michael G.;Kastead, Kyle A.;Postle, Kathleen
通讯作者：
Postle, Kathleen
影响因子：
3.4
作者：
KUWAHARA, M;VERKMAN, AS
通讯作者：
VERKMAN, AS
DOI：
10.1016/j.jasms.2006.06.006
发表时间：
2006-11-01
影响因子：
3.2
作者：
Englander, S. Walter
通讯作者：
Englander, S. Walter
影响因子：
48
作者：
Masson, Glenn R.;Burke, John E.;Rand, Kasper D.
通讯作者：
Rand, Kasper D.
影响因子：
56.9
作者：
Shultis, David D.;Purdy, Michael D.;Wiener, Michael C.
通讯作者：
Wiener, Michael C.