Quantifying the insertion of membrane proteins into lipid bilayer nanodiscs using a fusion protein strategy.
Quantifying the insertion of membrane proteins into lipid bilayer nanodiscs using a fusion protein strategy.
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使用融合蛋白策略量化膜蛋白插入脂质双层纳米盘的情况。
DOI：
10.1016/j.bbamem.2020.183190
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
F. Hagn
中科院分区：
文献类型：
--
作者：
Elisabeth Häusler;K. Fredriksson;I. Goba;Carsten Peters;K. Raltchev;L. Sperl;A. Steiner;S. Weinkauf;F. Hagn
膜蛋白的寡聚状态调节其在各种细胞过程中的功能。由于膜蛋白必须溶解在适当的膜模拟物中，因此使用经典的生物物理方法来分析蛋白质寡聚体是具有挑战性的。我们在这里提出了一种方法来确定插入到脂质纳米盘的膜蛋白的数量。它是基于选择性地定量小而稳健的融合蛋白的量的能力，所述融合蛋白可以在掺入脂质纳米盘后从膜蛋白上蛋白水解裂解。在确定的组装条件下，每个纳米盘的膜蛋白数量的详细知识对于估计寡聚化的趋势是必不可少的，而且对于指导需要存在同质寡聚状态的结构研究的样品优化也是必不可少的。我们表明，这种方法可以有效地用于确定在各种组装条件下的纳米盘中的VDAC 1通道的数量，证实了负染色EM。所提出的方法特别适用于膜蛋白，不能很容易地通过其他方法，如单跨度跨膜螺旋探测。该测定可应用于可并入纳米盘中而不需要特殊仪器的任何膜蛋白，因此将广泛适用于量化脂质纳米盘中的膜蛋白插入的其他方法并与之互补。
A membrane protein's oligomeric state modulates its functionality in various cellular processes. Since membrane proteins have to be solubilized in an appropriate membrane mimetic, the use of classical biophysical methods to analyze protein oligomers is challenging. We here present a method to determine the number of membrane proteins inserted into lipid nanodiscs. It is based on the ability to selectively quantify the amount of a small and robust fusion protein that can be proteolytically cleaved off from a membrane protein after incorporation into lipid nanodiscs. A detailed knowledge of the number of membrane proteins per nanodisc at defined assembly conditions is essential to estimate the tendency for oligomerization, but also for guiding sample optimization for structural investigations that require the presence of a homogenous oligomeric state. We show that this method can efficiently be used to determine the number of VDAC1 channels in nanodiscs at various assembly conditions, as confirmed by negative stain EM. The presented method is suitable in particular for membrane proteins that cannot be probed easily by other methods such as single span transmembrane helices. This assay can be applied to any membrane protein that can be incorporated into a nanodisc without the requirement for special instrumentation and will thus be widely applicable and complementary to other methods that quantify membrane protein insertion in lipid nanodiscs.
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影响因子：
3
作者：
Tang, Guang;Peng, Liwei;Ludtke, Steven J.
通讯作者：
Ludtke, Steven J.
影响因子：
--
作者：
Ritchie, T. K.;Grinkova, Y. V.;Bayburt, T. H.;Denisov, I. G.;Zolnerciks, J. K.;Atkins, W. M.;Sligar, S. G.
通讯作者：
Sligar, S. G.
影响因子：
15
作者：
Denisov, IG;Grinkova, YV;Sligar, SG
通讯作者：
Sligar, SG
DOI：
10.1016/s0021-9258(18)42569-0
发表时间：
1992-04
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Mark A. LemmonS;J. Flanagan;J. Hunt;B. Adair;Barbara-Jean Bormanng;Christopher E. Dempseyll;D. Engelman
通讯作者：
Mark A. LemmonS;J. Flanagan;J. Hunt;B. Adair;Barbara-Jean Bormanng;Christopher E. Dempseyll;D. Engelman
影响因子：
64.8
作者：
Molina, Daniel Martinez;Wetterholm, Anders;Nordlund, Paer
通讯作者：
Nordlund, Paer