Rapid corepressor exchange from the trp-repressor/operator complex: An NMR study of [ul-13C/15N]-l-tryptophan

Weontae Lee, Matthew Revington, Neil A. Farrow, Asao Nakamura, Naoko Utsunomiya-Tate, Yoko Miyake, Masatsune Kainosho, Cheryl H. Arrowsmith

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Abstract

[ul-13C/15N]-l-tryptophan was prepared biosynthetically and its dynamic properties and intermolecular interaction with a complex of Escherichia coli trp-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 15N-1H HSQC, 13C-1H HSQC, 13C-and 15N-edited 2D NOESY spectra. The exchange off-rate of the corepressor between the bound and free states was determined to be 3.4±0.52 s-1 at 45°C, almost three orders of magnitude faster than the dissociation of the protein-DNA complex. Examination of the experimental NOE buildup curves indicates that it may be desirable to use longer mixing times than would normally be used for a large molecule, in order to detect weak intermolecular NOEs in the presence of exchange. Intermolecular NOEs from bound corepressor to trp-repressor and DNA were analyzed with respect to the mechanism of ligand exchange. This analysis suggests that, in order for the ligand to diffuse out of the complex, there must be significant movement or 'breathing' of the protein and/or DNA.

Original languageEnglish
Pages (from-to)367-375
Number of pages9
JournalJournal of Biomolecular NMR
Volume5
Issue number4
DOIs
Publication statusPublished - 1995 Jun 1

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Spectroscopy

Cite this

Lee, W., Revington, M., Farrow, N. A., Nakamura, A., Utsunomiya-Tate, N., Miyake, Y., Kainosho, M., & Arrowsmith, C. H. (1995). Rapid corepressor exchange from the trp-repressor/operator complex: An NMR study of [ul-13C/15N]-l-tryptophan. Journal of Biomolecular NMR, 5(4), 367-375. https://doi.org/10.1007/BF00182280