A T1 rho-filtered two-dimensional transferred NOE spectrum for studying antibody interactions with peptide antigens.
Transferred nuclear Overhauser effect (TRNOE) spectroscopy can be used to study intra- and intermolecular interactions of bound ligands complexed with large proteins. However, the 2D NOE (NOESY) spectra of large proteins are very poorly resolved and it is very difficult to discriminate the TRNOE cross peaks, especially those due to intermolecular interactions, from the numerous cross peaks due to intramolecular interactions in the protein. In previous studies we measured two-dimensional difference spectra that show exclusively TRNOE and exchange cross-peaks (Anglister, J., 1990. Quart. Rev. Biophys. 23:175-203). Here we show that a filtering method based on the difference between the T1rho values of the ligand and the protein protons can be used to directly obtain a two-dimensional transferred NOE spectrum in which the background cross-peaks due to intramolecular interactions in the protein are very effectively removed. The usefulness of this technique to study protein ligand interactions is demonstrated for two different antibodies complexed with a peptide of cholera toxin (CTP3). It is shown that the T1 rho-filtering alleviates t problems encountered in our previous measurements of TRNOE by the difference method. These problems were due to imperfections in the subtraction of two spectra measured for two different samples.
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
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Scherf
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Anglister
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000737:chemistry; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002772:Cholera Toxin / Q000737:chemistry / Q000276:immunology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011522:Protons
D000911:Antibodies, Monoclonal; D000941:Antigens; D010446:Peptide Fragments; D011522:Protons; C050310:cholera toxin, B subunit (50-64); D002772:Cholera Toxin