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

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Summary

[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.

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Abbreviations

NOESY:

nuclear Overhauser enhancement spectroscopy

HSQC:

heteronuclear single-quantum coherence

PFG:

pulsed field gradient

l-Trp:

l-tryptophan

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Authors and Affiliations

  1. Division of Molecular and Structural Biology, Ontario Cancer Institute, University of Toronto, 500 Sherbourne Street, M4X 1K9, Toronto, Ontario, Canada

    Weontae Lee, Matthew Revington & Cheryl H. Arrowsmith

  2. Department of Medical Biophysics, University of Toronto, 500 Sherbourne Street, M4X 1K9, Toronto, Ontario, Canada

    Weontae Lee, Matthew Revington & Cheryl H. Arrowsmith

  3. Department of Molecular and Medical Genetics, University of Toronto, M5S 1A8, Toronto, Ontario, Canada

    Neil A. Farrow

  4. Central Research Laboratories, Ajinomoto Co. Inc., 1-1 Suzuki-cho, 210, Kawasaki, Japan

    Asao Nakamura

  5. Department of Chemistry, Tokyo Metropolitan University, 1-1 Minamiohsawa, 192-03, Hachioji, Japan

    Naoko Utsunomiya-Tate, Yoko Miyake & Masatsune Kainosho

Authors
  1. Weontae Lee
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  2. Matthew Revington
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  3. Neil A. Farrow
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  4. Asao Nakamura
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  5. Naoko Utsunomiya-Tate
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  6. Yoko Miyake
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  7. Masatsune Kainosho
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  8. Cheryl H. Arrowsmith
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Lee, W., Revington, M., Farrow, N.A. et al. Rapid corepressor exchange from the trp-repressor/operator complex: An NMR study of [ul-13C/15N]-l-tryptophan. J Biomol NMR 5, 367–375 (1995). https://doi.org/10.1007/BF00182280

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  • DOI: https://doi.org/10.1007/BF00182280

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