Site-directed mutagenesis and 1H nuclear magnetic resonance of an anti-dinitrophenyl spin label antibody.
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-SL 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-Gly2). AN02 mutants with approximately 50 and 200-fold enhanced affinities for DNP-Gly2 are engineered. In these mutants, a single amino acid change relieves an electrostatic interaction between DNP-Gly2 and the binding site. The resulting improvement in hapten binding ability is specific to DNP-Gly2, since the affinity for DNP-SL is relatively unchanged. Evidence of conformational heterogeneity in the AN02/DNP-Gly2 complex is presented. In contrast with DNP-Gly, a ring proton of DNP-Gly2 experiences two environments on binding to AN02. It was previously shown that a light chain tyrosine (LY31) also assumes two conformations when DNP-Gly2 is bound. The simultaneous presence of multiple AN02/DNP-Gly2 complexes implies conformational isomerism of a tryptophan residue which contacts both the hapten and LY31.
Department of Chemistry, Stanford University, CA 94305.
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Martinez-Yamout
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McConnell
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D016466:CHO Cells; D006224:Cricetinae; D004140:Dinitrophenols / Q000276:immunology*; D006241:Haptens; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013113:Spin Labels
D000911:Antibodies, Monoclonal; D004140:Dinitrophenols; D006241:Haptens; D013113:Spin Labels