Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR.
Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR.
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DOI：
10.1007/s10858-015-9903-1
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发表时间：
2015-04
影响因子：
2.7
通讯作者：
Griffin, Robert G.
中科院分区：
文献类型：
--
作者：
Eddy, Matthew T.;Su, Yongchao;Silvers, Robert;Andreas, Loren;Clark, Lindsay;Wagner, Gerhard;Pintacuda, Guido;Emsley, Lyndon;Griffin, Robert G.
人类电压依赖性阴离子通道1（VDAC）是一种32 kDa的β-桶整合膜蛋白，控制离子跨线粒体外膜的转运。尽管确定了VDAC解决方案和衍射结构，但其功能机制的结构基础尚未完全理解。生物物理学研究表明，VDAC需要一个脂质双层来实现全部功能，从而激发了对膜环境中VDAC原子分辨率结构信息的需求。在这里，我们报告了实现这一目标的重要一步：通过魔角自旋核磁共振（MAS NMR）在DMPC脂质双层中广泛分配VDAC的主链和侧链共振。VDAC重组到DMPC脂质双层自发形成2维脂质晶体，显示出显著的光谱分辨率（在750 MHz下，13 C线宽为0.5-0.3 ppm，15 N线宽小于0.5 ppm）。除了在脂质双层中工作的益处之外，还观察到脂质结晶制剂的几个明显优点。首先，强信号和尖锐的线宽促进了通过MAS NMR对脂质双层中的完整膜β-桶蛋白的广泛NMR共振分配。第二，环区中的大量残基容易观察到并分配，这在洗涤剂溶解的膜蛋白中可能是具有挑战性的，其中环区由于构象交换的谱线加宽而通常检测不到。第三，完整的主链和侧链化学位移分配可以获得的前25个残基，其中包括功能上重要的N-末端。所报告的分配使我们能够比较DMPC 2D脂质晶体、DMPC脂质体和LDAO溶解样品中制备的VDAC的预测扭转角，以解决膜模拟环境对蛋白质构象的可能影响。最后，我们讨论了报告的分配方法的优点和缺点，以及通过1H检测MAS NMR进行更完整的分配研究和从头结构测定的巨大潜力。
The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms 2-dimensional lipid crystals, showing remarkable spectral resolution (0.5–0.3 ppm for 13C line width and less than 0.5 ppm 15N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via 1H detected MAS NMR.
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影响因子：
5.6
作者：
Bhate MP;Wylie BJ;Tian L;McDermott AE
通讯作者：
McDermott AE
影响因子：
4.4
作者：
BENNETT, AE;RIENSTRA, CM;GRIFFIN, RG
通讯作者：
GRIFFIN, RG
影响因子：
1.7
作者：
Baldus, M;Petkova, AT;Griffin, RG
通讯作者：
Griffin, RG
DOI：
10.1073/pnas.051629298
发表时间：
2001-02-27
影响因子：
11.1
作者：
Fernández, C;Adeishvili, K;Wüthrich, K
通讯作者：
Wüthrich, K
DOI：
10.1073/pnas.0900908106
发表时间：
2009-06-09
影响因子：
11.1
作者：
Bajaj, Vikram S.;Mak-Jurkauskas, Melody L.;Griffin, Robert G.
通讯作者：
Griffin, Robert G.