Nuclear magnetic resonance studies of flexible opiate conformations at monoclonal antibody binding sites. Quantitative interproton distances obtained from comparing theoretical and experimental transferred nuclear Overhauser enhancement: correlation with antibody sequence.
A previous publication described the use of qualitative intramolecular 1H-transferred nuclear Overhauser effect measurements to determine the conformations of flexible ligands at monoclonal anti-opiate antibody binding sites. This paper concentrates on the quantitative interpretation of experiments of this type using the ligand nalorphine (N-allyl morphine) and a single anti-opiate monoclonal antibody. I compare the experimental unidimensional driven nuclear Overhauser effect buildup curves to theoretical curves derived with a knowledge of the fixed interproton distances in the ligand. The discussion covers the potential accuracies of derived distances and concentrates on two problem areas associated with determining structures from this type of experiment. The most serious one is the case where, because of particular multiproton spatial distributions, spin diffusion is so rapid that it cannot be determined experimentally and where numerical fits of theoretical calculations are misleading. The results show that, while intraligand spin diffusion complicates the interpretation for some proton pairs, with many others accuracies within about 0.3 A for interproton distances from 2 to 4 A are attainable. The results confirm the earlier report that the conformation of nalorphine in this antibody binding site differs from the major one present in solution or in the crystal. An important aspect of the work is that theoretical prediction of nuclear Overhauser effect time-dependence is an important practical tool for recognizing cases where interpretation of experiments will be difficult. Initial data on protein-to-ligand transferred nuclear Overhauser effect are presented, which show that at least one aromatic amino acid residue is closely involved in the binding of the ligand. The companion paper presents the primary sequences of the variable regions of the antibodies being used in our studies. In this paper, these and associated immunochemical studies are correlated with the nuclear magnetic resonance results. The combination of data presented in the two papers provides a basis for future work on protein-ligand interproton distances in the range 1 to 5 A using both transferred nuclear Overhauser effect (for rapidly exchanging ligands) and isotope-edited, indirectly detected nuclear Overhauser effect (for tightly bound ligands).
Glasel
J A
JA
Department of Biochemistry, University of Connecticut Health Center, Farmington 06032.
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001666:Binding Sites, Antibody; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D009682:Magnetic Resonance Spectroscopy; D009269:Nalorphine / Q000032:analysis* / Q000378:metabolism; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D009269:Nalorphine