SITE-DIRECTED MUTAGENESIS AND H-1 NUCLEAR-MAGNETIC-RESONANCE OF AN ANTI-DINITROPHENYL SPIN-LABEL ANTIBODY

[摘要] Mutagenesis and spin label difference spectroscopy are used to assign the resonances of tyrosine residues in the binding site of the anti-dinitrophenyl spin label (DNP-SL) antibody AN02. Hapten binding constants of 13 point mutants are determined. From these studies it is clear that a light chain tyrosine specifically stabilizes DNP-BL binding, perhaps by means of a hydrogen bond to the hapten. This bond is absent in the case of the diamagnetic hapten dinitrophenyl-diglycine (DNP-Gly(2)). AN02 mutants with similar to 50 and 200-fold enhanced affinities for DNP-Gly(2) are engineered. In these mutants, a single amino acid change relieves an electrostatic interaction between DNP-Gly(2) and the binding site. The resulting improvement in hapten binding ability is specific to DNP-Gly(2), since the affinity for DNP-SL is relatively unchanged. Evidence of conformational heterogeneity in the AN02/DNP-Gly(2) complex is presented. In contrast with DNP-Gly, a ring proton of DNP-Gly(2), experiences two environments on binding to AN02. It was previously shown that a light chain tyrosine (LY31) also assumes two conformations when DNP-Gly(2) is bound. The simultaneous presence of multiple ANOS/DNP-Gly(2) complexes implies conformational isomerism of a tryptophan residue which contacts both the hapten and LY31.

[发布日期] 1994-12-02

[关键词] MUTAGENESIS;NMR;ANTIBODY;SPIN LABEL HAPTEN;AFFINITY ENHANCEMENT