Structure determination of a membrane protein in proteoliposomes.
Structure determination of a membrane protein in proteoliposomes.
复制标题
DOI：
10.1021/ja209464f
复制
发表时间：
2012-02-01
影响因子：
15
通讯作者：
Opella, Stanley J.
中科院分区：
文献类型：
--
作者：
Das, Bibhuti B.;Nothnagel, Henry J.;Lu, George J.;Son, Woo Sung;Tian, Ye;Marassi, Francesca M.;Opella, Stanley J.
通过测定汞转运体MerF的60个残基螺旋-环-螺旋整体膜核MerFt的结构，证明了一种确定蛋白脂质体中膜蛋白三维结构的核磁共振方法。该方法结合了取向样品（OS）固态核磁共振和魔角旋转（MAS）固态核磁共振技术，测量了在无取向液晶磷脂双层中均匀13C/15N标记蛋白的单个残基相对于单个外轴（双层法向）的取向限制。该方法依靠膜蛋白在双层中的快速（> - 105hz）旋转扩散，将静态化学位移各向异性和异核偶极-偶极耦合粉末模式平均为轴对称粉末模式，减少了频率范围。与这种运动平均粉末图案的平行边缘相关的频率与从宏观上平行于施加磁场方向的固定样品光谱中的单线共振测量的频率完全相同。所有数据都是在接受MAS的无取向样本上收集的。通过MAS对样品的同核13C/13C偶极偶联进行平均，可以使用均匀的13C/15N标记蛋白质，通过直接13C检测以及使用多维MAS固态核磁共振方法来解析和分配共振，从而提高灵敏度。该方法的独特之处在于取向约束的测量，这使得在无取向的蛋白脂质体中可以确定蛋白质的结构和取向。
An NMR method for determining the three-dimensional structures of membrane proteins in proteoliposomes is demonstrated by determining the structure of MerFt, the 60-residue helix–loop–helix integral membrane core of the 81-residue mercury transporter MerF. The method merges elements of oriented sample (OS) solid-state NMR and magic angle spinning (MAS) solid-state NMR techniques to measure orientation restraints relative to a single external axis (the bilayer normal) from individual residues in a uniformly 13C/15N labeled protein in unoriented liquid crystalline phospholipid bilayers. The method relies on the fast (>105 Hz) rotational diffusion of membrane proteins in bilayers to average the static chemical shift anisotropy and heteronuclear dipole–dipole coupling powder patterns to axially symmetric powder patterns with reduced frequency spans. The frequency associated with the parallel edge of such motionally averaged powder patterns is exactly the same as that measured from the single line resonance in the spectrum of a stationary sample that is macroscopically aligned parallel to the direction of the applied magnetic field. All data are collected on unoriented samples undergoing MAS. Averaging of the homonuclear 13C/13C dipolar couplings, by MAS of the sample, enables the use of uniformly 13C/15N labeled proteins, which provides enhanced sensitivity through direct 13C detection as well as the use of multidimensional MAS solid-state NMR methods for resolving and assigning resonances. The unique feature of this method is the measurement of orientation restraints that enable the protein structure and orientation to be determined in unoriented proteoliposomes.
登录
查看更多内容
影响因子：
1.7
作者：
Baldus, M;Petkova, AT;Griffin, RG
通讯作者：
Griffin, RG
影响因子：
4.4
作者：
Chan, JCC;Tycko, R
通讯作者：
Tycko, R
影响因子：
15
作者：
De Angelis, AA;Nevzorov, AA;Opella, SJ
通讯作者：
Opella, SJ
影响因子：
2.2
作者：
Filipp, Fabian V.;Sinha, Neeraj;Opella, Stanley J.
通讯作者：
Opella, Stanley J.
影响因子：
5.6
作者：
Cady, Sarah D.;Mishanina, Tatiana V.;Hong, Me
通讯作者：
Hong, Me