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- Title
Improved NMR spectra of a protein–DNA complex through rational mutagenesis and the application of a sensitivity optimized isotope-filtered NOESY experiment.
- Authors
Iwahara, Junji; Wojciak, Jonathan M.; Clubb, Robert T.
- Abstract
The NMR spectra of the complex between the DNA-binding domain of the Dead ringer protein (DRI-DBD, Gly262-Gly398) and its DNA binding site (DRI-DBD:DNA, 26 kDa) have been optimized by biochemical and spectroscopic means. First, we demonstrate the utility of a modified 2D [F1,F2] 13C-filtered NOESY experiment that employs a 1JHC versus chemical shift optimized adiabatic 13C inversion pulse [Zwahlen, C. et al. (1997) J. Am. Chem. Soc., 119, 6711–6721]. The new sequence is shown to be more sensitive than previously published pulse schemes (up to 40% in favorable cases) and its utility is demonstrated using two protein–DNA complexes. Second, we demonstrate that the targeted replacement of an interfacial aromatic residue in the DRI-DBD:DNA complex substantially reduces line broadening within its NMR spectra. The spectral changes are dramatic, salvaging a protein–DNA complex that was originally ill suited for structural analysis by NMR. This biochemical approach is not a general method, but may prove useful in the spectral optimization of other protein complexes that suffer from interfacial line broadening caused by dynamic changes in proximal aromatic rings.
- Subjects
NUCLEIC acids spectra; DNA; GENES; SPECTRUM analysis; NUCLEIC acids; MUTAGENESIS
- Publication
Journal of Biomolecular NMR, 2001, Vol 19, Issue 3, p231
- ISSN
0925-2738
- Publication type
Article
- DOI
10.1023/A:1011296112710