Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated alpha-chain from human hemoglobin A.
Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated alpha-chain from human hemoglobin A.
复制标题
溶液 1H NMR 研究人血红蛋白 A 中分离的 α 链氰基复合物的活性位点分子结构和磁性。
DOI：
10.1016/s1570-9639(03)00202-4
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
LaMar,GerdN
中科院分区：
文献类型：
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作者：
Tran,Anh-TuyetT;Kolczak,Urszula;LaMar,GerdN
用同核二维1H核磁共振研究了分离的人成人血红蛋白α链的血红素单体复合体的溶液电子结构和分子结构，以便建立一种二聚链的指认方案，以指导完整的异四聚体复合体的类似指认，并比较α链及其亚单位的结构。目标残基是那些表现出显著(|0.2|ppm)偶极移位的残基，正如根据一小部分容易分配的活性中心残基确定的一组初步的磁轴所预测的那样。所有97个靶残基(占总∼的70%)都是利用多肽主干的“初步”磁轴预测的温度依赖性来分配的；它们包括所有被认为在调节四聚体HbA的配基亲和力方面发挥重要作用的残基。未分配的是偶极位移小于|0.2|ppm的A-螺旋、G-螺旋末端和H-螺旋起点。完整的赋值允许确定一组稳健的顺磁磁化率张量的取向和各向异性，从而根据连接的α-亚单位的晶体坐标定量解释α-链的偶极位移，这反过来证实了分离的α-链相对于完整的HBA有一个非常保守的分子结构。与Fe-CN单元倾斜相关的长磁轴与血红素法线倾斜10°，因此Fe-CN单元向∼-β-H倾斜的方式与HbACO中的Fe-CO倾斜非常相似。结论是，一组“初步的”磁轴和变温二维核磁共振谱的使用对于有效地指定花环α链是至关重要的，这种方法在血红蛋白中也应该是同样有效的。
The solution electronic and molecular structure for the heme pocket of the cyanomet complex of the isolated α-chain of human adult hemoglobin (HbA) has been investigated by homonuclear two-dimensional1H NMR in order to establish an assignment protocol for the dimeric chain that will guide similar assignments in the intact, heterotetrameric HbA complex, and to compare the structures of the α-chain with its subunit in HbA. The target residues are those that exhibit significant (>|0.2| ppm) dipolar shifts, as predicted by a “preliminary” set of magnetic axes determined from a small set of easily assigned active site residues. All 97 target residues (∼70% of total) were assigned by taking advantage of the temperature dependence predicted by the “preliminary” magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than |0.2| ppm. The complete assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the α-chain in terms of the crystal coordinates of the α-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated α-chain relative to that in the intact HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted ∼10° from the heme normal so that the Fe-CN unit is tilted toward the β-meso-H in a fashion remarkably similar to the Fe-CO tilt in HbACO. It is concluded that a set of “preliminary” magnetic axes and the use of variable temperature two-dimensional NMR spectra are crucial to effective assignments in the cyanomet α-chain and that this approach should be similarly effective in HbA.