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Title: Structural studies of DNP-binding immunoglobulins
Author: Jackson, Ronald C. J.
ISNI:       0000 0001 3588 1686
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1979
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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 VL 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 VL 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 VL dimer is described. The proposed site is a large cavity bounded by the aromatic side-chains of the Trp-93L and Tyr-34L 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 VL 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-34L has no effect on the affinity of protein 315 or of the VL dimer for several ligands. It is concluded that no hydrogen bond is formed between the phenolic group of Tyr-34L and the 2-nitro group of the ligand. From measurements of the perturbation of the visible absorption spectrum of protein 315 nitrated at Tyr-33H, 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.
Supervisor: Dwek, Raymond A. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Immunoglobulins ; Tryptophan