Efficient and stable reconstitution of the ABC transporter BmrA for solid-state NMR studies.
Efficient and stable reconstitution of the ABC transporter BmrA for solid-state NMR studies.
复制标题
用于固态NMR研究的ABC转运蛋白BMRA的有效稳定重建。
DOI：
10.3389/fmolb.2014.00005
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发表时间：
2014
影响因子：
5
通讯作者：
Böckmann A
中科院分区：
文献类型：
--
作者：
Kunert B;Gardiennet C;Lacabanne D;Calles-Garcia D;Falson P;Jault JM;Meier BH;Penin F;Böckmann A
我们提出了枯草芽孢杆菌atp结合盒（ABC）转运体BmrA的固态核磁共振样品制备和第一个二维光谱，BmrA是一种参与多药耐药的膜蛋白。同型二聚体130-kDa蛋白由于其膜结合性质、大小、固有的柔韧性和不溶性，对结构表征是一个挑战。我们发现，在枯草芽孢杆菌的脂质中，以0.5 w/w的脂蛋白比重构这种蛋白质，可以在脂质膜中实现最佳的蛋白质插入，同时满足两个核心核磁共振要求，光谱中的高信噪比和样品在数月时间内的稳定性。从窄的共振线和BmrA二级结构典型的信号色散可以看出，所获得的光谱指向折叠良好的蛋白质和高度均匀的制备。这为研究转运体在输出周期中的不同构象状态，以及通过化学位移指纹和顺序共振分配研究与底物的相互作用开辟了道路。
We present solid-state NMR sample preparation and first 2D spectra of the Bacillus subtilis ATP-binding cassette (ABC) transporter BmrA, a membrane protein involved in multidrug resistance. The homodimeric 130-kDa protein is a challenge for structural characterization due to its membrane-bound nature, size, inherent flexibility and insolubility. We show that reconstitution of this protein in lipids from Bacillus subtilis at a lipid-protein ratio of 0.5 w/w allows for optimal protein insertion in lipid membranes with respect to two central NMR requirements, high signal-to-noise in the spectra and sample stability over a time period of months. The obtained spectra point to a well-folded protein and a highly homogenous preparation, as witnessed by the narrow resonance lines and the signal dispersion typical for the expected secondary structure distribution of BmrA. This opens the way for studies of the different conformational states of the transporter in the export cycle, as well as on interactions with substrates, via chemical-shift fingerprints and sequential resonance assignments.