Rapid corepressor exchange from the trp-repressor/operator complex: an NMR study of [ul-13C/15N]-L-tryptophan.
[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 degrees 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.
Lee
W
W
Division of Molecular and Structural Biology, Ontario Cancer Institute, University of Toronto, Canada.
Revington
M
M
Farrow
N A
NA
Nakamura
A
A
Utsunomiya-Tate
N
N
Miyake
Y
Y
Kainosho
M
M
Arrowsmith
C H
CH
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002247:Carbon Isotopes; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D008024:Ligands; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D009875:Operator Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics*; D014364:Tryptophan / Q000737:chemistry*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D004269:DNA, Bacterial; D008024:Ligands; D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins