Proton positions in the Mn2+ binding site of concanavalin A as determined by single-crystal high-field ENDOR spectroscopy
Proton positions in the Mn2+ binding site of concanavalin A as determined by single-crystal high-field ENDOR spectroscopy
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DOI：
10.1021/ja0104305
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发表时间：
2001-08-29
影响因子：
15
通讯作者：
Goldfarb, D
中科院分区：
文献类型：
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作者：
Carmieli, R;Manikandan, P;Goldfarb, D
高场(95 GHz)脉冲EPR和电子-核双共振(Endor)技术首次被用于确定蛋白质单晶中高自旋金属中心的配位体质子坐标。刀豆蛋白A含有一个与两个水分子配位的Mn2+离子、一个组氨酸残基和三个羧酸盐。在H2O和D2O中生长刀豆蛋白A单晶，以区分可交换的水质子和咪唑基的非可交换质子。通过在EPR谱内的不同磁场下进行Endor测量，选择了不同的EPR转变。这种选择，再加上4.5K时获得的巨大热极化和类似于3.4T的磁场，使我们能够将Endor信号分配给它们各自的ms流形，从而提供了超精细耦合的迹象。在Ac晶面和ab晶面获得了旋转图。在每个平面上确定了两个不同的晶位，并通过模拟旋转图确定了其中两个咪唑质子和四个水质子的超精细张量。所有质子都具有轴对称的超精细张量，并且通过应用点-偶极近似，确定了各种质子相对于Mn2+离子的位置。同样，利用已公布的蛋白质的超高分辨率X射线晶体坐标，确定了参与氢键与邻近残基的水质子。
High-field (95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) techniques have been used for the first time to determine coordinates of ligand protons of a high-spin metal center in a protein single crystal. The protein concanavalin A contains a Mn2+ ion which is coordinated to two water molecules, a histidine residue, and three carboxylates. Single crystals of concanavalin A were grown in H2O and in D2O to distinguish the exchangeable water protons from the nonexchangeable protons of the imidazole group. Distinct EPR transitions were selected by performing the ENDOR measurements at different magnetic fields within the EPR spectrum. This selection, combined with the large thermal polarization achieved at 4.5 K and a magnetic field of similar to3.4 T allowed us to assign the ENDOR signals to their respective Ms manifolds, thus providing the signs of the hyperfine couplings. Rotation patterns were acquired in the ac and ab crystallographic planes. Two distinct crystallographic sites were identified in each plane, and the hyperfine tensors of two of the imidazole protons and the four water protons were determined by simulations of the rotation patterns. All protons have axially symmetric hyperfine tensors and, by applying the point-dipole approximation, the positions of the various protons relative to the Mn2+ ion were determined. Likewise, the water protons involved in H-bonding to neighboring residues were identified using the published, ultrahigh-resolution X-ray crystallographic coordinates of the protein.