Improved NMR spectra of a protein-DNA complex through rational mutagenesis and the application of a sensitivity optimized isotope-filtered NOESY experiment.
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 1J(HC) 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.
Iwahara
J
J
Department of Chemistry and Biochemistry and UCLA-DOE Laboratory of Structural Biology and Genetics, University of California at Los Angeles, 90095, USA.
Wojciak
J M
JM
Clubb
R T
RT
D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; C098342:Retn protein, Drosophila; D004247:DNA