Proton-n.m.r. study of interaction of myelin basic protein with a monoclonal antibody

Abstract

Proton n.m.r. at 400 MHz has been applied to study the interactions of bovine or porcine myelin basic protein (b- or p-MBP) with a monoclonal antibody to human (h-) MBP. The antibody, an IgG immunoglobulin that contains a sequential epitopic region, cross-reacts with b-MBP but not with p-MBP, the presumed epitope being identical in h- and b-MBP. N.m.r. spectra were recorded from the Fab fragment of the antibody and for mixtures of Fab and MBP at various molar ratios. The n.m.r. spectrum of MBP in the mixture consists mostly of well resolved peaks against a broad background due to the Fab. With b-MBP, but not p-MBP, specific interactions are observed at the residue tyrosine-135, which is part of the epitopic sequence. Other interactions occur between the Fab and both b- and p-MBP at residues distant from the epitopic region. Standard radioassay techniques were employed to calculate the binding constants of both basic proteins with the immunoglobulin. The binding constant, Kb, for IgG to column-immobilized b-MBP at 298K is (0.95 +/- 0.07) X 10(7) dm3/mol. The value of Kb decreases with the ionic strength of the medium, suggesting a coulombic interaction between antigen and antibody. N.m.r. spectra were also measured for mixtures of the Fab fragment and peptides containing the epitopic site, with results in agreement with those for the whole protein.

Interactions of myelin basic protein with mixed dodecylphosphocholine/palmitoyllysophosphatidic acid micelles

“…In the segment comprising residues 120-140, A-133 and S-137 in r-MBP are substituted by proline residues in p-MBP. Variations in the conformation of this region of MBP from different species were established in NMR studies of the interactions of the protein with a monoclonal antibody to an epitope consisting of the linear sequence of residues 130-139 of bovine MBP (Mendz et al, 1985). The fact that some resonances of peptide 99-179 " Segments predicted to be capable of forming amphipathic helices are shown.…”

The behavior of aged regenerated Bombyx mori silk fibroin solutions studied by 1H NMR and rheology

“…Therefore, the 1 H resonances of the RSF aqueous solution prior to gelation have been assigned by closely examining the expanded 1 H NMR spectra (Fig. 2a-f) and matching their values with the values of all amino acid residues available in the literature [60][61][62][63][64][65][66], as summarized in Table 1. Taking into account that both fresh and aged silk solution strongly demonstrated the presence of mixed conformations, so the 1 H resonances were determined as an average of all resonances emerged from random coil, a-helix and b-strand.…”

N.m.r. studies of myelin basic protein. Conformation of a peptide that is an antigenic determinant for B-cell reactivity

“…The conformation of the octapeptide, as revealed by this n.m.r. study in aqueous solution, may be compared with the interaction of the epitopic region of MBP with the monoclonal antibody (Mendz et al, 1985). The position of the tyrosine residue is consistent with its important role in binding to the receptor site.…”

“…A 'H n.m.r. study of the interaction of b-MBP with one of the monoclonal antibodies showed a strong effect at the tyrosine residue (Tyr-1 35) in the basic protein, but no effect at the histidine residue (His-139) (Mendz et al, 1985). A preliminary n.m.r.…”