Characterization of a site-directed mutant of cytochrome b5 designed to alter axial imidazole ligand plane orientation.
Characterization of a site-directed mutant of cytochrome b5 designed to alter axial imidazole ligand plane orientation.
复制标题
细胞色素 b5 定点突变体的表征，旨在改变轴向咪唑配体平面方向。
DOI：
10.1021/bi961858x
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Guiles,RD
中科院分区：
文献类型：
--
作者：
Sarma,S;Dangi,B;Yan,C;DiGate,RJ;Banville,DL;Guiles,RD
细胞色素B5突变体的设计，以实现重新定位的个别轴向咪唑配体。轴向配体平面的取向被认为调节双（咪唑）轴向连接血红素蛋白的还原电位。A67V突变通过在空间位阻疏水血红素结合口袋中用更大的疏水配体取代残基来实现这一目标。使用从1H和15N 2D同向和异向NMR光谱和1H −15N 3D异向NMR光谱获得的限制计算突变区域中突变和野生型蛋白质的溶液结构。在两种结构的改进中使用每个残基超过10个限制。20个精细结构的平均局部rmsd对于野生型结构为0.30 μ m，对于A67V突变体为0.38 μ m。通过还原蛋白质的15N −1H杂原子相关光谱的叠加，实现了酰胺质子共振归属从野生型到突变蛋白质的转移。使用常规分配策略建立侧链分配和顺序分配。使用与应用于野生型蛋白质的程序类似的方法计算各向异性顺磁磁化率张量分量的方向，表明野生型和突变蛋白质中平面内分量的方向是相同的。然而，计算的突变体蛋白质的磁化率张量的z分量的方向与计算的野生型蛋白质的方向相比，A型的差异为17°，B型的差异为11°。磁化率张量的z分量的旋转（朝向δ meso质子）与突变引起的H63咪唑环的旋转方向相同，并且大小相同。
Mutants of cytochromeb5were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V mutation achieved this goal through the substitution of a bulkier, hydrophobic ligand for a residue, in the sterically hindered hydrophobic heme binding pocket. Solution structures of mutant and wild-type proteins in the region of the mutation were calculated using restraints obtained from1H and15N 2D homonuclear and heteronuclear NMR spectra and1H−15N 3D heteronuclear NMR spectra.. More than 10 restraints per residue were used in the refinement of both structures. Average local rmsd for 20 refined structures was 0.30 Å for the wild-type structure and 0.38 Å for the A67V mutant. The transfer of amide proton resonance assignments from wild-type to the mutant protein was achieved through overlays of15N−1H heteronuclear correlation spectra of the reduced proteins. Side chain assignments and sequential assignments were established using conventional assignment strategies. Calculation of the orientation of the components of the anisotropic paramagnetic susceptibility tensor, using methods similar to procedures applied to the wild-type protein, shows that the orientation of the in-plane components are identical in the wild-type and mutant proteins. However, the orientation of the z-component of the susceptibility tensor calculated for the mutant protein differs by 17° for the A-form and by 11° for the B-form from the orientation calculated for the wild-type protein. The rotation of thez-component of the susceptibility tensor (toward the δ meso proton) is in the same direction and is of the same magnitude as the rotation of the H63 imidazole ring induced by mutation.