Structural studies of DNP-binding immunoglobulins

• Main Author: Jackson, Ronald C. J.
• Other Authors: Dwek, Raymond A.
• Published: University of Oxford 1979
• Subjects: 547.7; Immunoglobulins : Tryptophan
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.460573

The importance of tryptophan in the combining sites of anti-DNP antibodies is evaluated from a series of model studies. The thermodynamic parameters characterizing the formation of a DNP/tryptophan complex are determined. The contribution of this interaction to the affinity and specificity of anti-DNP antibodies is discussed. The accuracy of antibody combining site structures generated by model-building is examined, using available crystallographic data. The average error of alpha-carbon atom positions is estimated to be 1-2 Å. The binding of nitrophenyl compounds to the V_L dimer of the DNP-binding mouse myeloma protein 315 is investigated by ¹H n.m.r. It is concluded that any corformational changes are small, and that the physical basis for the DNP-binding specificity of the V_L dimer is the conservation of structural features which are important in determining the specificity of the intact protein 315. A model of the combining site of the V_L dimer is described. The proposed site is a large cavity bounded by the aromatic side-chains of the Trp-93_L and Tyr-34_L residues. The structure is able to explain the large upfield chemical shift changes of the ligand resonances observed on binding. The kinetic parameters and structural extent of the pH-dependent conformational change of the V_L dimer are investigated by fluorescence and ¹H n.m.r. The transition does not obey a reversible one-step mechanism, and is limited in extent. The involvement of two tyrosine residues in the hypervariable regions of protein 315 is investigated by specific nitration. Nitration of Tyr-34_L has no effect on the affinity of protein 315 or of the V_L dimer for several ligands. It is concluded that no hydrogen bond is formed between the phenolic group of Tyr-34_L and the 2-nitro group of the ligand. From measurements of the perturbation of the visible absorption spectrum of protein 315 nitrated at Tyr-33_H, it is concluded that this residue is in proximity to the side-chains, but not the nitrophenyl rings, of bound ligands. Several aspects of the interaction of a homogeneous mouse anti-DNP antibody, protein A3, with nitrophenyl and similar ligands are described. The findings are discussed in relation to the heterogeneity and cross-reactivity of antisera.