title: Interactions of antibody aromatic residues with a peptide of cholera toxin observed by two-dimensional transferred nuclear Overhauser effect difference spectroscopy.
abstract: The interactions between a peptide of cholera toxin and the aromatic amino acids of the TE33 antipeptide antibody, cross-reactive with the toxin, have been studied by NOESY difference spectroscopy. The 2D difference between the NOESY spectrum of the Fab with a 4-fold excess of the peptide and that of the peptide-saturated Fab reveals cross-peaks growing with excess of the peptide. These cross-peaks are due to magnetization transfer between the Fab and neighboring bound peptide protons, and a further transfer to the free peptide protons by exchange between bound and free peptide (transferred NOE). Additional cross-peaks appearing in the difference spectrum are due to a combination of intramolecular interactions between bound peptide protons and exchange between bound and free peptide. Assignment of cross-peaks is attained by specific deuteration of antibody aromatic amino acids using also the resonance assignment of the free peptide, deduced from the COSY spectrum of the peptide solution. The antibody combining site is found to be highly aromatic. We have identified one or two histidine, two tyrosine, and two tryptophan residues and one phenylalanine residue of the antibody interacting with valine-3, proline-4, glycine-5, glutamine-7, histidine-8, and aspartate-10 of the peptide. The 2D TRNOE difference spectroscopy can be used to study protein-ligand interactions, given that the ligand off rate is fast relative to the spin-lattice relaxation time of the protein and ligand protons (about 1 s). The resolution obtained in the difference spectra implies that the technique is equally applicable for studying proteins having a molecular weight larger than 50,000.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Polymer Research, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: J
authors.initials: J
authors.lastname: Anglister
authors.forename: R
authors.initials: R
authors.lastname: Levy
authors.forename: T
authors.initials: T
authors.lastname: Scherf
mesh_terms: D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D002772:Cholera Toxin / Q000276:immunology*; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D010649:Phenylalanine; D014364:Tryptophan
chemical_list: D010446:Peptide Fragments; D010649:Phenylalanine; D014364:Tryptophan; D002772:Cholera Toxin