Tran Anh-Tuyet T, Kolczak Urszula, La Mar Gerd N
Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
Biochim Biophys Acta. 2003 Aug 21;1650(1-2):59-72. doi: 10.1016/s1570-9639(03)00202-4.
The solution electronic and molecular structure for the heme pocket of the cyanomet complex of the isolated alpha-chain of human adult hemoglobin (HbA) has been investigated by homonuclear two-dimensional 1H 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 alpha-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 (approximately 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 alpha-chain in terms of the crystal coordinates of the alpha-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated alpha-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 approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-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 alpha-chain and that this approach should be similarly effective in HbA.
为了建立二聚体链的归属方案，以指导完整的异源四聚体血红蛋白A（HbA）复合物中的类似归属，并比较α链与其在HbA中的亚基结构，通过同核二维¹H NMR研究了成人人类血红蛋白（HbA）分离的α链的氰化高铁复合物血红素口袋的溶液电子和分子结构。目标残基是那些表现出显著（>0.2 ppm）偶极位移的残基，这是根据从一小部分易于归属的活性位点残基确定的“初步”磁轴集预测的。利用“初步”磁轴预测的多肽主链的温度依赖性，对所有97个目标残基（约占总数的70%）进行了归属；它们包括所有被认为在调节四聚体HbA中配体亲和力方面起重要作用的残基。未被归属的是A螺旋、G螺旋末端和H螺旋起始处，其偶极位移小于0.2 ppm。完整的归属使得能够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HbA中α亚基的晶体坐标对α链的偶极位移进行定量解释，这反过来又证实了分离的α链相对于完整HbA的分子结构在很大程度上是保守的。与Fe-CN单元倾斜相关的主要磁轴相对于血红素法线倾斜约10度，使得Fe-CN单元以与HbACO中Fe-CO倾斜非常相似的方式向β-间位-H倾斜。得出的结论是，一组“初步”磁轴和可变温度二维NMR光谱的使用对于氰化高铁α链的有效归属至关重要，并且这种方法在HbA中应该同样有效。
