Dower S K, Wain-Hobson S, Gettins P, Givol D, Jackson W R, Perkins S J, Sunderland C A, Sutton B J, Wright C E, Dwek R A
Biochem J. 1977 Aug 1;165(2):207-23. doi: 10.1042/bj1650207.
Magnetic-resonance techniques are used to refine the model of the combining site of the Fv fragment of the dinitrophenyl-binding mouse myeloma protein MOPC 315 constructed by Padlan, Davies, Pecht, Givol & Wright (1976) (Cold Spring Harbor Symp. Quant. Biol.41, in the press). Light-absorption studies indicate a dinitrophenyl-tryptophan interaction in the Fv fragment of the type occurring in free solution. The Dnp-aspartate-tryptophan complex is therefore used as a starting point for the n.m.r. (nuclear-magnetic-resonance) analysis of the dinitrophenyl-Fv fragment interaction. Ring-current calculations are used to determine the geometry of the complex. The specificity of complex-formation between dinitrophenyl and tryptophan is confirmed by the lack of ring-current shifts of the dinitrophenyl resonances when tryptophan is replaced by any other aromatic amino acid. Proton n.m.r. difference spectra (at 270MHz), resulting from the addition of a variety of haptens to the Fv fragment, show that the combining site is highly aromatic in nature. Calculations on the basis of ring-current shifts define the geometry of the combining site, which involves a dinitrophenyl ring in van der Waals contact with four aromatic amino acid residues on the protein. The observation of a nuclear Overhauser effect on the H((3)) resonance of the dinitrophenyl ring provides additional constraints on the relative geometry of the H((3)) proton and an aromatic amino acid residue on the Fv fragment. The specificity of the Fv fragment for dinitrophenyl ligands arises from a stacking interaction of the dinitrophenyl ring with tryptophan-93(L), in an ;aromatic box' of essentially tryptophan-93(L), phenylalanine-34(H) and tyrosine-34(L); asparagine-36(L) and tyrosine-34(L) also contribute by forming hydrogen bonds with the nitro groups on the dinitrophenyl ring. The n.m.r. results also confirm that the antibody-hapten reaction may be visualized as a single encounter step. An Appendix shows the method of calculation of ring currents for the four aromatic amino acids and their use in calculating structures.
磁共振技术被用于完善由帕德兰、戴维斯、佩希特、吉沃尔和赖特（1976年）构建的二硝基苯基结合小鼠骨髓瘤蛋白MOPC 315的Fv片段结合位点模型（《冷泉港定量生物学研讨会论文集》41卷，即将出版）。光吸收研究表明，在Fv片段中存在自由溶液中那种类型的二硝基苯基 - 色氨酸相互作用。因此，二硝基苯 - 天冬氨酸 - 色氨酸复合物被用作核磁共振分析二硝基苯基 - Fv片段相互作用的起点。利用环电流计算来确定复合物的几何结构。当色氨酸被任何其他芳香族氨基酸取代时，二硝基苯基共振没有环电流位移，这证实了二硝基苯基与色氨酸之间形成复合物的特异性。向Fv片段中添加各种半抗原后得到的质子核磁共振差异光谱（在270MHz）表明，结合位点本质上是高度芳香性的。基于环电流位移的计算确定了结合位点的几何结构，该结构涉及一个与蛋白质上四个芳香族氨基酸残基处于范德华接触的二硝基苯环。对二硝基苯环的H((3))共振观察到核Overhauser效应，这为H((3))质子与Fv片段上一个芳香族氨基酸残基的相对几何结构提供了额外的限制条件。Fv片段对二硝基苯基配体的特异性源于二硝基苯环与色氨酸 - 93(L)在一个基本上由色氨酸 - 93(L)、苯丙氨酸 - 34(H)和酪氨酸 - 34(L)组成的“芳香盒”中的堆积相互作用；天冬酰胺 - 36(L)和酪氨酸 - 34(L)也通过与二硝基苯环上的硝基形成氢键而做出贡献。核磁共振结果还证实，抗体 - 半抗原反应可以被视为一个单步相遇过程。附录展示了四种芳香族氨基酸环电流的计算方法及其在结构计算中的应用。
