Solution nuclear magnetic resonance determination of active site structure for a paramagnetic protein: cyanomet Aplysia myoglobin.
Solution nuclear magnetic resonance determination of active site structure for a paramagnetic protein: cyanomet Aplysia myoglobin.
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溶液核磁共振测定顺磁性蛋白的活性位点结构：cyanomet 海兔肌红蛋白。
DOI：
10.1006/jmbi.1993.1348
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发表时间：
1993
影响因子：
5.6
通讯作者：
Brunori,M
中科院分区：
文献类型：
--
作者：
Qin,J;LaMar,GN;Ascoli,F;Brunori,M
本文用核磁共振法测定了一种顺磁性蛋白质-青蟹肌红蛋白（Mb）在溶液中的活性部位结构，以探讨这种缺乏组氨酸残基的蛋白质的配体稳定机制。结构的确定依赖于使用实验的偶极位移从二维NMR分配，NOE和顺磁诱导弛豫作为约束条件与磁化率张量的组合。磁化率张量是通过使用高分辨率的晶体坐标的其他衍生物的AfrussiaMb（特别是metMb，metMbF和metMbN 3）的分子的一部分，这是不受扰动的不同的配体，连同观察到的偶极位移afrussiametMbCN，寻找欧拉旋转，正确地转换晶体坐标的磁轴。通过使用基于三组晶体坐标的保守结构部分的各种输入数据集，可以很好地定义磁各向异性以及磁轴的取向。骨架部分（E、F、G螺旋段和FG角）和近端侧链的计算位移与观测位移之间的拟合很好，这表明AmmonsiametMbCN的骨架部分和近端侧链的溶液结构与晶体学研究的其他三种衍生物相同。另一方面，远端侧被证明是目前较小的，但重要的结构变化，相对于AstradiametMbF，这反映了结合的双原子配体（CN-），而不是一个单原子。特别地，Arg E10（66）的胍基被重新定位，并且进一步远离铁，其距离与结合氰化物的氢键一致。结合的氰化物与血红素垂直方向倾斜约108 °（通过主磁轴检测），大约朝向γ-meso-H。这种倾斜似乎是由于与Arg E10（66）的吸引氢键和来自Ile E11（67）的空间排斥。结果强烈支持的作用，精氨酸E10在稳定的阴离子配体的氢键之前提出的，并证明了有效性的偶极位移作为一个独特的和敏感的约束，在确定低自旋铁态的顺磁性蛋白质的活性位点结构。
The active site structure of a paramagnetic protein, cyanomet myoglobin (Mb) fromAplysia limacina, has been determined in solution by NMR in order to investigate the mechanism of ligand stabilization in this protein, which lacks the usual distal His residue. The structure determination relies on using the experimental dipolar shifts from two-dimensional NMR assignments, NOEs and paramagnetic-induced relaxations as the constraints with a combination of the magnetic susceptibility tensor. The magnetic susceptibility tensor was obtained by using the high resolution crystal co-ordinates of other derivatives ofAplysiaMb (notably metMb, metMbF and metMbN3) for the part of the molecule which is unperturbed by the different ligands, together with the observed dipolar shifts ofAplysiametMbCN, to search for the Euler rotation that correctly converts the crystal co-ordinates to the magnetic axes. The magnetic anisotropies, as well as the orientation of the magnetic axes, are well defined by using the various input data sets based on the conserved structural portions of three sets of crystal co-ordinates. An excellent fit between the calculatedversusthe observed shifts was obtained for both backbone protions (E, F, G helical segments and FG corner) and the proximal side-chains, which demonstrates that the solution structure of the backbone portions and the proximal side forAplysiametMbCN is identical to those for three other derivatives investigated by crystallography. On the other hand, the distal side was shown to present minor but important structural changes relative toAplysiametMbF, which reflect the binding of a diatomic ligand (CN-) rather than a monoatomic one. In particular, the guanidinium group of Arg E10(66) is repositioned and further away from the iron at a distance consistent with H-bonding to the bound cyanide. The bound cyanide is titled ∼8° (as detected by the major magnetic axis) away from the heme normal approximately towards the γ-meso-H. This tilt appears to be due to the attractive hydrogen-bonding with Arg E10(66) and the steric repulsion from Ile E11(67). The results strongly support the role of Arg E10 in stabilizing the anionic ligands by hydrogen bonding as proposed before and demonstrate the validity of the dipolar shifts as a unique and sensitive constraint in determining the active site structure of paramagnetic proteins in the low-spin ferric state.