Sarma S, Dangi B, Yan C, DiGate R J, Banville D L, Guiles R D
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore 21201, USA.
Biochemistry. 1997 May 13;36(19):5645-57. doi: 10.1021/bi961858x.
Mutants of cytochrome b5 were 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 from 1H and 15N 2D homonuclear and heteronuclear NMR spectra and 1H-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 A for the wild-type structure and 0.38 A for the A67V mutant. The transfer of amide proton resonance assignments from wild-type to the mutant protein was achieved through overlays of 15N-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 degrees for the A-form and by 11 degrees for the B-form from the orientation calculated for the wild-type protein. The rotation of the z-component of the susceptibility tensor (toward the delta meso proton) is in the same direction and is of the same magnitude as the rotation of the H63 imidazole ring induced by mutation.
细胞色素b5的突变体旨在实现单个轴向咪唑配体的重新定向。轴向配体平面的取向被认为可调节轴向连接双（咪唑）血红素蛋白的还原电位。A67V突变通过在空间位阻较大的疏水血红素结合口袋中，用一个更大的疏水配体取代一个残基实现了这一目标。利用从1H和15N二维同核和异核NMR谱以及1H-15N三维异核NMR谱获得的约束条件，计算了突变区域内突变体和野生型蛋白的溶液结构。在两种结构的优化中，每个残基使用了超过10个约束条件。20个优化结构的平均局部均方根偏差，野生型结构为0.30 Å，A67V突变体为0.38 Å。通过还原蛋白的15N-1H异核相关谱的叠加，实现了酰胺质子共振归属从野生型蛋白到突变体蛋白的转移。使用传统的归属策略建立了侧链归属和序列归属。使用与应用于野生型蛋白的程序类似的方法，计算各向异性顺磁磁化率张量各分量的取向，结果表明，野生型和突变体蛋白中平面内分量的取向相同。然而，计算得到的突变体蛋白的磁化率张量z分量的取向，与野生型蛋白相比，A形式相差17度，B形式相差11度。磁化率张量z分量的旋转（朝向δ中位质子）方向相同，且与突变诱导的H63咪唑环的旋转幅度相同。
