RAPID COREPRESSOR EXCHANGE FROM THE TRP-REPRESSOR OPERATOR COMPLEX - AN NMR-STUDY OF [UL-C-13/N-15]-L-TRYPTOPHAN/

[ul-C-13/N-15]-L-tryptophan was prepared biosynthetically and its dyna mic properties and intermolecular interaction with a complex of Escher ichia coli tip-repressor and a 20 base-pair operator DNA were studied by heteronuclear isotope-edited NMR experiments. The resonances of the free and bound corepressor (L-Trp) were unambiguously identified from gradient-enhanced N-15-H-1 HSQC, C-13-H-1 HSQC, C-13- and N-15-edited 2D NOESY spectra. The exchange off-rate of the corepressor between th e bound and free states was determined to be 3.4 +/- 0.52 s(-1) at 45 degrees C, almost three orders of magnitude faster than the dissociati on of the protein-DNA complex. Examination of the experimental NOE bui ldup curves indicates that it may be desirable to use longer mixing ti mes than would normally be used for a large molecule, in order to dete ct weak intermolecular NOEs in the presence of exchange. Intermolecula r NOEs from bound corepressor to trp-repressor and DNA were analyzed w ith respect to the mechanism of ligand exchange. This analysis suggest s that, in order for the ligand to diffuse out of the complex, there m ust be significant movement or 'breathing' of the protein and/or DNA.