Structure refinement and membrane positioning of selectively labeled OmpX in phospholipid nanodiscs.
Structure refinement and membrane positioning of selectively labeled OmpX in phospholipid nanodiscs.
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DOI：
10.1007/s10858-014-9883-6
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发表时间：
2015-04
影响因子：
2.7
通讯作者：
Wagner, Gerhard
中科院分区：
文献类型：
--
作者：
Hagn, Franz;Wagner, Gerhard
考虑到膜模拟物的贡献，膜蛋白的核磁共振结构研究往往由于其大的尺寸而变得复杂。因此，经典的共振分配方法常常失败。大尺寸的磷脂纳米盘，一种无洗涤剂的磷脂双层模拟物，到目前为止阻碍了它们在高分辨率溶液状态核磁共振光谱中的应用。我们最近推出了适合于核磁共振结构测定的更小的纳米盘。然而，纳米盘中膜蛋白的侧链分配仍然难以捉摸。在这里，我们利用基于NOE的方法来分配(立体)标记的Ile、Leu、Val和Ala甲基标记的以及均匀标记的15N-Phe和15N-Tyr标记的OmpX，并计算了精细化的高分辨率结构。此外，我们使用PF1噬菌体介质诱导弱对齐，获得了OmpX在纳米盘中的残留偶极耦合(RDC)。将得到的核磁共振结构的反计算NOESY谱与实验NOESYs进行比较，以验证这些结构的质量。我们进一步利用质子化的脂头基团和侧链甲基之间的NOE信息来确定OmpX在磷脂双层中的位置。这些数据被用Gd3+修饰的脂类进行的顺磁松弛增强(Pre)实验所证实。综上所述，这项研究强调了在高氚背景下对膜蛋白进行(立体)特异性标记以进行高分辨率结构测定的必要性，特别是在磷脂纳米盘等大型膜模拟系统中。通过NOESY反计算进行结构验证将有助于膜蛋白的结构确定和确认，而膜蛋白的NOE分配往往是困难的。将蛋白质用于脂类NON将有利于膜蛋白在脂双层中的定位，而不需要制备多个蛋白质样品。
NMR structural studies on membrane proteins are often complicated by their large size, taking into account the contribution of the membrane mimetic. Therefore, classical resonance assignment approaches often fail. The large size of phospholipid nanodiscs, a detergent-free phospholipid bilayer mimetic, prevented their use in high-resolution solution-state NMR spectroscopy so far. We recently introduced smaller nanodiscs that are suitable for NMR structure determination. However, side-chain assignments of a membrane protein in nanodiscs still remain elusive. Here, we utilized a NOE-based approach to assign (stereo-) specifically labeled Ile, Leu, Val and Ala methyl labeled and uniformly 15N-Phe and 15N-Tyr labeled OmpX and calculated a refined high-resolution structure. In addition, we were able to obtain residual dipolar couplings (RDCs) of OmpX in nanodiscs using Pf1 phage medium for the induction of weak alignment. Back-calculated NOESY spectra of the obtained NMR structures were compared to experimental NOESYs in order to validate the quality of these structures. We further used NOE information between protonated lipid head groups and side-chain methyls to determine the position of OmpX in the phospholipid bilayer. These data were verified by paramagnetic relaxation enhancement (PRE) experiments obtained with Gd3+-modified lipids. Taken together, this study emphasizes the need for the (stereo-) specific labeling of membrane proteins in a highly deuterated background for high-resolution structure determination, particularly in large membrane mimicking systems like phospholipid nanodiscs. Structure validation by NOESY back-calculation will be helpful for the structure determination and validation of membrane proteins where NOE assignment is often difficult. The use of protein to lipid NOEs will be beneficial for the positioning of a membrane protein in the lipid bilayer without the need for preparing multiple protein samples.
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影响因子：
15
作者：
Hyberts, Sven G.;Takeuchi, Koh;Wagner, Gerhard
通讯作者：
Wagner, Gerhard
影响因子：
2.7
作者：
Diercks, T;Coles, M;Kessler, H
通讯作者：
Kessler, H
影响因子：
2.7
作者：
Ayala, Isabel;Sounier, Remy;Boisbouvier, Jerome
通讯作者：
Boisbouvier, Jerome
影响因子：
2.7
作者：
DELAGLIO, F;GRZESIEK, S;BAX, A
通讯作者：
BAX, A
影响因子：
2.7
作者：
Plevin, Michael J.;Hamelin, Olivier;Gans, Pierre
通讯作者：
Gans, Pierre